WT1 antigen peptide conjugate vaccine

ABSTRACT

A compound represented by the formula (1): 
                         
wherein X a  and Y a  are each a single bond and the like, cancer antigen peptide A is an MHC class I-restricted WT1 peptide consisting of 7-30 amino acid residues, R 1  is a hydrogen atom, a group represented by the formula (2):
 
                         
wherein X b  and Y b  are each a single bond and the like, cancer antigen peptide B has a sequence different from that of the cancer antigen peptide A, and is an MHC class I-restricted WT1 peptide consisting of 7-30 amino acid residues, or cancer antigen peptide C, and cancer antigen peptide C has a sequence different from that of the cancer antigen peptide A, and is an MHC class I-restricted WT1 peptide or an MHC class II-restricted WT1 peptide, consisting of 7-30 amino acid residues containing one cysteine residue, or a salt thereof, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of and claims the benefits of priority to U.S. Ser.No. 14/549,091, filed Nov. 20, 2014, which is a continuation ofInternational Application No. PCT/JP2014/059336, filed Mar. 28, 2014,which claims the benefits of priority to Japanese Application No.2013-072173, filed Mar. 29, 2013 and Japanese Application No.2013-158383, filed Jul. 31, 2013. The entire contents of all of theabove applications are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

1. Technical Field

The present invention belongs to the field of cancer immunotherapy, andrelates to a conjugate vaccine that can be subjected to trimming bypeptidase ERAP1, is obtained by conjugating peptide precursors derivedfrom WT1 antigen protein via a sulfur-sulfur covalent bond, andefficiently induces cytotoxic T cells.

2. Background Art

For eradication of cancer cells in the body, cellular immunity,particularly cytotoxic T cell (cytotoxic T-lymphocyte, Cytotoxic T-cell,hereinafter to be referred to as CTL) mainly plays an important role.CTL is produced by differentiation and proliferation of a precursor Tcell that recognized a complex formed by an antigen peptide derived froma cancer antigen protein (cancer antigen peptide) and an MHC class Imolecule, and attacks cancer cells.

A cancer suppressor gene of Wilms tumor, WT1 (WT1 gene) is considered anew cancer antigen protein for leukemia and solid tumor (see non-patentdocument 1).

As for WT1 protein, for example, the following cancer antigen peptidesthat are bound to and presented by MHC class I have been reported (seepatent documents 1, 2).

WT1₁₂₆₋₁₃₄ peptide: RMFPNAPYL (Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu) (SEQID NO: 2),

WT1₂₃₅₋₂₄₃ peptide: CMTWNQMNL (Cys-Met-Thr-Trp-Asn-Gln-Met-Asn-Leu) (SEQID NO: 3),

WT1₁₀₋₁₈ peptide: ALLPAVPSL (Ala-Leu-Leu-Pro-Ala-Val-Pro-Ser-Leu) (SEQID NO: 5),

WT1₁₈₇₋₁₉₅ peptide: SLGEQQYSV (Ser-Leu-Gly-Glu-Gln-Gln-Tyr-Ser-Val) (SEQID NO: 6),

WT1₃₀₂₋₃₁₀ peptide: RVPGVAPTL (Arg-Val-Pro-Gly-Val-Ala-Pro-Thr-Leu) (SEQID NO: 7) and the like.

In cancer immunotherapy, activation of helper T cell is also importantfor activating other T cells including CTL. In general, an antigenprotein is degraded by intracellular lysosome, a part of peptidefragments constituted by a peptide consisting of about 13-17 amino acidresidues binds as an antigen peptide to MHC class II molecule and ispresented to helper T cell-TCR•CD3 complex to activate helper T cell. Asfor WT1 protein, for example, the following cancer antigen peptides thatare presented by binding to MHC class II have been reported (see patentdocuments 3-5).

WT1₃₃₂₋₃₄₇ peptide: KRYFKLSHLQMHSRKH(Lys-Arg-Tyr-Phe-Lys-Leu-Ser-His-Leu-Gln-Met-His-Ser-Arg-Lys-His) (SEQID NO: 8), WT1₃₂₈₋₃₄₉ peptide: PGCNKRYFKLSHLQMHSRKHTG(Pro-Gly-Cys-Asn-Lys-Arg-Tyr-Phe-Lys-Leu-Ser-His-Leu-Gln-Met-His-Ser-Arg-Lys-His-Thr-Gly)(SEQ ID NO: 10),WT1₁₂₂₋₁₄₀ peptide: SGQARMFPNAPYLPSCLES(Ser-Gly-Gln-Ala-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-Pro-Ser-Cys-Leu-Glu-Ser)(SEQ ID NO: 11) and the like.

As a vaccine antigen of WT1, an antigen protein itself or theaforementioned antigen protein-derived antigen peptide is mainly used(see non-patent document 2). Since a cancer vaccine using a proteingenerally contains various cancer antigen peptides, it cansimultaneously induce a plurality of CTLs and helper T cells. On theother hand, since the cancer protein vaccine possesses problems instable supply and quality control, peptides that facilitate productionand quality control are widely used as cancer antigen of WT1. Generally,however, since conventional peptide vaccines are mainly constituted by asingle MHC class I-presented peptide antigen, it has been pointed out inrecent years that efficient induction of CTL requires furtherimprovement (see non-patent document 3).

One of the solving means is a multivalent antigen peptide presenting WT1peptide cancer vaccine. As such peptide cancer vaccine, a cocktailvaccine containing a mixture of a plurality of peptide antigenspresented by MHC class I and class II (see non-patent document 4), along chain peptide vaccine containing peptide antigens presented by MHCclass I and class II, which are bound by an amide bond, and the likehave been reported (see non-patent document 5). In the case of acocktail vaccine, however, since each peptide antigen composed ofvarious amino acids shows various properties, the development of anoptimal formulation capable of efficiently inducing CTL correspondingthereto is often problematic. In the case of a long chain peptidevaccine, the production thereof sometimes poses problems, like protein.Furthermore, since peptide antigens presented by class I and class IIare bonded via any peptide spacer in a long chain peptide vaccine,control and prediction of the cleavage sites by intracellular enzyme aredifficult. In the meantime, a peptide dimer wherein two peptide monomersare mutually bonded by a disulfide bond has been reported (see patentdocument 6). Different from cocktail vaccine, two single peptides arebonded, and therefore, they have single physical property and can beproduced conveniently. On the other hand, to form a conjugate, WT1cancer antigen peptides are required to contain cysteine in the aminoacid sequence thereof, and therefore, applicable ones are limited.Furthermore, an altered compound obtained by condensing the N-terminalcysteine of cancer antigen peptide with cysteine, glutathione orthioglycolic acid by a disulfide bond has also been reported (see patentdocument 7).

The process of cancer antigen peptide presentation on MHC class Iinvolves a plurality of peptidases. Of such peptidases, Endoplasmicreticulum aminopeptidase 1 (hereinafter to be referred to as ERAP1) isone of the trimming enzymes in the endoplasmic reticulum (hereinafter tobe referred to as ER), and has been reported to recognize a particularantigen peptide sequence and the peptide length, and cleaves the cancerantigen peptide precursor from the N-terminal to control the length tobe optimal for binding to MHC class I (see non-patent documents 6-8).However, there is no report to date on a WT1 peptide cancer antigenprecursor containing cysteine, that is controlled in its length from theN-terminal, by the trimming function of ERAP1.

DOCUMENT LIST Patent Documents

-   patent document 1: WO 00/06602-   patent document 2: WO 00/18795-   patent document 3: WO 2005/045027-   patent document 4: WO 2007/047764-   patent document 5: WO 2007/120673-   patent document 6: WO 2004/063217-   patent document 7: WO 2007/063903

Non-Patent Documents

-   non-patent document 1: The Journal of Immunology, 2000; 164(4);    1873-1880-   non-patent document 2: The Oncologist, 2012; 17(2); 250-259-   non-patent document 3: Cancer Journal, 2011; 17(5); 343-350-   non-patent document 4: Blood, 2010; 166(2); 171-179-   non-patent document 5: Cancers, 2011; 3; 3991-4009-   non-patent document 6: Proceedings of the National Academy of    Sciences of United States of America, 2005; 102(47); 17107-17112-   non-patent document 7: The Journal of Immunology, 2009; 183;    5526-5536-   non-patent document 8: The Journal of Immunology, 2010; 184;    4725-4732

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The problem to be solved by the present invention is to provide a WT1conjugate vaccine that induces CTL efficiently.

Means of Solving the Problems

The present inventors have conducted intensive studies in an attempt tosolve the aforementioned problem, and conceived, when consideringadopting conjugate vaccine, an idea of adding cysteine in WT1 cancerantigen peptide, and further confirmed that the results ofpharmacological tests and the like using in vivo animal model stronglysuggest that ERAP1 cleaves cysteine from the N-terminal of a WT1 cancerantigen peptide precursor that is generated by intracellular reductivecleavage of disulfide bond, to efficiently convert the same to a cancerantigen peptide, which in turn led to the finding of a polyvalentantigen peptide presenting conjugate vaccine capable of inducing CTL inthe body, and the completion of the present invention.

To be specific, during the process of studying the solving means to theabove-mentioned problem, they have obtained an idea of a method forintroducing cysteine, which is necessary for forming a conjugate of twodifferent WT1 cancer antigen peptides, into any position of theN-terminal or C-terminal, without influencing the antigen presentationby MHC class I. As a result of further study, they have created apeptide by introducing 0-5 amino acids containing cysteine into the Nterminal of a WT1 cancer antigen peptide, and a conjugate of thepeptides containing a disulfide bond via cysteine. Furthermore, thepresent inventors have confirmed for the first time that said peptideand the conjugate are susceptible to trimming by ERAP1 in vitro and/orin vivo, which in turn results in the formation of a cancer antigenpeptide, and thereby, completed the present invention.

While the development of a novel multivalent WT1 antigen peptidepresenting peptide cancer vaccine, which can be produced easily, isapplicable to various ones, and induces CTL with high efficiency, hasbeen desired, the conjugate invented by the present inventors hasenabled the development of a WT1 conjugate vaccine that induces CTLefficiently, is superior in physicochemical properties, can be producedeasily, facilitates production management, and is applicable to variousones.

Accordingly, the present invention relates to the following.

1. First Embodiment

1. A compound represented by the formula (1):

wherein X^(a) and Y^(a) are each independently a single bond or adivalent peptide group consisting of 1-4 amino acid residues, and atotal of the amino acid residue number for X^(a) and the amino acidresidue number for Y^(a) is an integer of 0-4, cancer antigen peptide Ais an MHC class I-restricted WT1 peptide consisting of 7-30 amino acidresidues, an amino group of an N-terminal amino acid of the cancerantigen peptide A binds to Y^(a) in the formula (1), and a carbonylgroup of a C-terminal amino acid of the cancer antigen peptide A bindsto a hydroxyl group in the formula (1),R¹ is a hydrogen atom, a group represented by the formula (2):

wherein X^(b) and Y^(b) are each independently a single bond or adivalent peptide group consisting of 1-4 amino acid residues, and atotal of the amino acid residue number for X^(b) and the amino acidresidue number for Y^(b) is an integer of 0-4, cancer antigen peptide Bhas a sequence different from that of the cancer antigen peptide A, andis an MHC class I-restricted WT1 peptide consisting of 7-30 amino acidresidues, an amino group of an N-terminal amino acid of the cancerantigen peptide B binds to Y^(b) in the formula (2), and a carbonylgroup of a C-terminal amino acid of the cancer antigen peptide B bindsto a hydroxyl group in the formula (2), anda thioether group in the formula (2) binds to a thioether group in theformula (1),or cancer antigen peptide C,wherein the cancer antigen peptide C has a sequence different from thatof the cancer antigen peptide A, and is an MHC class I-restricted WT1peptide consisting of 7-30 amino acid residues containing one cysteineresidue or an MHC class II-restricted WT1 peptide consisting of 7-30amino acid residues containing one cysteine residue, and a thioethergroup of the cysteine residue of the cancer antigen peptide C binds to athioether group in the formula (1),provided when R¹ is a hydrogen atom, the sequence of a compoundrepresented by the formula (1) is not the same as the partial sequenceof a WT1 protein,or a pharmaceutically acceptable salt thereof;2. the compound according to 1, wherein X^(a) is a divalent peptidegroup consisting of 2 amino acid residues and Y^(a) is a single bond, orX^(a) and Y^(a) are each independently a divalent peptide groupconsisting of 1 amino acid residue, or X^(a) is a single bond and Y″ isa divalent peptide group consisting of 2 amino acid residues, or X^(a)is a divalent peptide group consisting of 1 amino acid residue and Y^(a)is a single bond, or X^(a) is a single bond and Y^(a) is a divalentpeptide group consisting of 1 amino acid residue, or X^(a) and Y^(a) areeach a single bond, or a pharmaceutically acceptable salt thereof;3. the compound according to 1 or 2, wherein X^(a) is a single bond, andY^(a) is a single bond, an alanine residue, a leucine residue or amethionine residue, or a pharmaceutically acceptable salt thereof;4. the compound according to 1 or 2, wherein X^(a) is a single bond or adivalent peptide group consisting of 1 amino acid residue, and Y^(a) isa single bond, or a pharmaceutically acceptable salt thereof;5. the compound according to any one of 1-4, wherein X^(a) and Y^(a) areeach a single bond, or a pharmaceutically acceptable salt thereof;6. the compound according to any one of 1-5, wherein the cancer antigenpeptide A is an MHC class I-restricted WT1 peptide consisting of 7-15amino acid residues, or a pharmaceutically acceptable salt thereof;7. the compound according to any one of 1-6, wherein the cancer antigenpeptide A is a peptide comprising any amino acid sequence selected fromthe following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) ALLPAVPSL,(SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) and RVPGVAPTL, (SEQ ID NO: 7)ora peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 2, 3, 5, 6 and 7 but containingalteration of amino acid residue(s), and having a CTL inductionactivity, or a pharmaceutically acceptable salt thereof;8. the compound according to any one of 1-7, wherein the cancer antigenpeptide A is a peptide consisting of any amino acid sequence selectedfrom the following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) CYTWNQMNL,(SEQ ID NO: 4) ALLPAVPSL, (SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) andRVPGVAPTL, (SEQ ID NO: 7)or a pharmaceutically acceptable salt thereof;9. the compound according to any one of 1-8, wherein R¹ is a hydrogenatom, or a pharmaceutically acceptable salt thereof;10. the compound according to any one of 1-9, wherein the compoundrepresented by the formula (1) is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

CRMFPNAPYL, (SEQ ID NO: 13) CCMTWNQMNL, (SEQ ID NO: 14) CCYTWNQMNL,(SEQ ID NO: 15) CALLPAVPSL, (SEQ ID NO: 16) CSLGEQQYSV (SEQ ID NO: 17)and CRVPGVAPTL, (SEQ ID NO: 18)or a pharmaceutically acceptable salt thereof;11. the compound according to any one of 1-8, wherein R¹ is a grouprepresented by the formula (2), or a pharmaceutically acceptable saltthereof;12. the compound according to any one of 1-8 and 11, wherein X^(b) is adivalent peptide group consisting of 2 amino acid residues and Y^(b) isa single bond, or X^(b) and Y^(b) are each independently a divalentpeptide group consisting of 1 amino acid residue, or X^(b) is a singlebond and Y^(b) is a divalent peptide group consisting of 2 amino acidresidues, or X^(b) is a divalent peptide group consisting of 1 aminoacid residue and Y^(b) is a single bond, or X^(b) is a single bond andY^(b) is a divalent peptide group consisting of 1 amino acid residue, orX^(b) and Y^(b) are each a single bond, or a pharmaceutically acceptablesalt thereof;13. the compound according to any one of 1-8 and 11-12, wherein X^(b) isa single bond, and Y^(b) is a single bond, an alanine residue, a leucineresidue or a methionine residue, or a pharmaceutically acceptable saltthereof;14. the compound according to any one of 1-8 and 11-12, wherein X^(b) isa single bond or a divalent peptide group consisting of 1 amino acidresidue, and Y^(b) is a single bond, or a pharmaceutically acceptablesalt thereof;15. the compound according to any one of 1-8 and 11-14, wherein X^(b)and Y^(b) are each a single bond, or a pharmaceutically acceptable saltthereof;16. the compound according to any one of 1-8 and 11-15, wherein thecancer antigen peptide B is an MHC class I-restricted WT1 peptideconsisting of 7-15 amino acid residues, or a pharmaceutically acceptablesalt thereof;17. the compound according to any one of 1-8 and 11-16, wherein thecancer antigen peptide B is a peptide comprising any amino acid sequenceselected from the following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) ALLPAVPSL,(SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) and RVPGVAPTL, (SEQ ID NO: 7)ora peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 2, 3, 5, 6 and 7 but containingalteration of amino acid residue(s), and having a CTL inductionactivity, or a pharmaceutically acceptable salt thereof;18. the compound according to any one of 1-8 and 11-17, wherein thecancer antigen peptide B is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) CYTWNQMNL,(SEQ ID NO: 4) ALLPAVPSL, (SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) andRVPGVAPTL, (SEQ ID NO: 7)or a pharmaceutically acceptable salt thereof;19. the compound according to any one of 1-8 and 11-18, wherein thecompound represented by the formula (1) is a compound represented by theformula (3):

wherein the bond between C and C is a disulfide bond, or apharmaceutically acceptable salt thereof;20. the compound according to any one of 1-8, wherein R¹ is cancerantigen peptide C, or a pharmaceutically acceptable salt thereof;21. the compound according to any one of 1-8 and 20, wherein the cancerantigen peptide C is an MHC class I-restricted WT1 peptide consisting of7-15 amino acid residues, or a pharmaceutically acceptable salt thereof;22. the compound according to any one of 1-8 and 20-21, wherein thecancer antigen peptide C is a peptide comprising the following aminoacid sequence:CMTWNQMNL (SEQ ID NO: 3), ora peptide comprising an altered amino acid sequence, which is the aminoacid sequence of SEQ ID NO: 3 but containing alteration of amino acidresidue(s), and having a CTL induction activity, or a pharmaceuticallyacceptable salt thereof;23. the compound according to any one of 1-8 and 20-22, wherein thecancer antigen peptide C is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL, (SEQ ID NO: 4)or a pharmaceutically acceptable salt thereof;24. the compound according to any one of 1-8 and 20-23, wherein thecompound represented by the formula (1) is a compound represented by theformula (4):

wherein the bond between C and C is a disulfide bond, or a compoundrepresented by the formula (5):

wherein the bond between C and C is a disulfide bond,or a pharmaceutically acceptable salt thereof;25. the compound according to any one of 1-8 and 20, wherein the cancerantigen peptide C is an MHC class II-restricted WT1 peptide consistingof 14-30 amino acid residues, or a pharmaceutically acceptable saltthereof;26. the compound according to any one of 1-8, 20 and 25, wherein thecancer antigen peptide C is a peptide comprising any amino acid sequenceselected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11)PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20) PGCNKRYFKLSHLQMHSRKH,(SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 10)CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH (SEQ ID NO: 23)and CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24)ora peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 10-11 and 19-24 but containingalteration of amino acid residue(s), and having a helper T cellinduction activity, or a pharmaceutically acceptable salt thereof;27. the compound according to any one of 1-8, 20 and 25-26, wherein thecancer antigen peptide C is a peptide consisting of an amino acidsequence selected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH(SEQ ID NO: 23) and CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24)or a pharmaceutically acceptable salt thereof;28. the compound according to any one of 1-8, 20 and 25-27, wherein thecompound represented by the formula (1) is a compound represented by theformula (6):

wherein the bond between C and C is a disulfide bond, a compoundrepresented by the formula (7):

wherein the bond between C and C is a disulfide bond, a compoundrepresented by the formula (8):

wherein the bond between C and C is a disulfide bond, or a compoundrepresented by the formula (9):

wherein the bond between C and C is a disulfide bond,or a pharmaceutically acceptable salt thereof;29. a pharmaceutical composition comprising the compound according toany one of 1-28, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier;30. the pharmaceutical composition according to 29, which is used as acancer vaccine;31. use of the compound according to any one of 1-28 or apharmaceutically acceptable salt thereof for the production of a cancervaccine;32. a method of treating or preventing cancer, comprising administeringa therapeutically or prophylactically effective amount of the compoundaccording to any one of 1-28 or a pharmaceutically acceptable saltthereof to a WT1 positive cancer patient in need thereof; and33. a method of obtaining two different MHC class I-restricted epitopes,or an MHC class I-restricted epitope and an MHC class II-restrictedepitope, comprising reacting the compound according to any one of 1-28or a pharmaceutically acceptable salt thereof with ERAP1.

2. Second embodiment

1. A compound represented by the formula (1):

wherein X^(a) and Y^(a) are each independently a single bond or adivalent peptide group consisting of 1-4 amino acid residues, and atotal of the amino acid residue number for X^(a) and the amino acidresidue number for Y^(a) is an integer of 0-4, cancer antigen peptide Ais an MHC class I-restricted WT1 peptide consisting of 7-30 amino acidresidues, an amino group of an N-terminal amino acid of the cancerantigen peptide A binds to Y^(a) in the formula (1), and a carbonylgroup of a C-terminal amino acid of the cancer antigen peptide A bindsto a hydroxyl group in the formula (1),R¹ is a hydrogen atom, a group represented by the formula (2):

wherein X^(b) and Y^(b) are each independently a single bond or adivalent peptide group consisting of 1-4 amino acid residues, and atotal of the amino acid residue number for X^(b) and the amino acidresidue number for Y^(b) is an integer of 0-4, cancer antigen peptide Bhas a sequence different from that of the cancer antigen peptide A inthe sequence and is an MHC class I-restricted WT1 peptide consisting of7-30 amino acid residues, an amino group of an N-terminal amino acid ofthe cancer antigen peptide B binds to Y^(b) in the formula (2), and acarbonyl group of a C-terminal amino acid of the cancer antigen peptideB binds to a hydroxyl group in the formula (2), and a thioether group inthe formula (2) binds to a thioether group in the formula (1),or cancer antigen peptide C,wherein the cancer antigen peptide C is an MHC class I-restricted WT1peptide different from the cancer antigen peptide A in the sequence andconsisting of 7-30 amino acid residues containing one cysteine residueor an MHC class II-restricted WT1 peptide consisting of 7-30 amino acidresidues containing one cysteine residue, and a thioether group of thecysteine residue of the cancer antigen peptide C binds to a thioethergroup in the formula (1),provided when R¹ is a hydrogen atom, the sequence of the compoundrepresented by the formula (1) is not the same as the partial sequenceof a WT1 protein,or a pharmaceutically acceptable salt thereof;2. the compound according to 1, wherein X^(a) is a divalent peptidegroup consisting of 2 amino acid residues and Y^(a) is a single bond, orX^(a) and Y^(a) are each independently a divalent peptide groupconsisting of 1 amino acid residue, or X^(a) is a single bond and Y^(a)is a divalent peptide group consisting of 2 amino acid residues, orX^(a) is a divalent peptide group consisting of 1 amino acid residue andY^(a) is a single bond, or X^(a) is a single bond and Y^(a) is adivalent peptide group consisting of 1 amino acid residue, or X^(a) andY^(a) are each a single bond, or a pharmaceutically acceptable saltthereof;3. the compound according to 1 or 2, wherein X^(a) is a single bond, andY^(a) is a single bond, an alanine residue, a leucine residue or amethionine residue, or a pharmaceutically acceptable salt thereof;4. the compound according to 1 or 2, wherein X^(a) is a single bond or adivalent peptide group consisting of 1 amino acid residue, and Y^(a) isa single bond, or a pharmaceutically acceptable salt thereof;5. the compound according to any one of 1-4, wherein X^(a) and Y^(a) areeach a single bond, or a pharmaceutically acceptable salt thereof;6. the compound according to any one of 1-5, wherein the cancer antigenpeptide A is an MHC class I-restricted WT1 peptide consisting of 7-15amino acid residues, or a pharmaceutically acceptable salt thereof;7. the compound according to any one of 1-6, wherein the cancer antigenpeptide A is a peptide comprising any amino acid sequence selected fromthe following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) ALLPAVPSL,(SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) and

RVPGVAPTL, (SEQ ID NO: 7)ora peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 2, 3, 5, 6 and 7 but containingalteration of amino acid residue(s), and having a CTL inductionactivity, or a pharmaceutically acceptable salt thereof;8. the compound according to any one of 1-7, wherein the cancer antigenpeptide A is a peptide consisting of any amino acid sequence selectedfrom the following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) CYTWNQMNL,(SEQ ID NO: 4) ALLPAVPSL, (SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) andRVPGVAPTL, (SEQ ID NO: 7)or a pharmaceutically acceptable salt thereof;9. the compound according to any one of 1-8, wherein R¹ is a hydrogenatom, or a pharmaceutically acceptable salt thereof;10. the compound according to any one of 1-9, wherein the compoundrepresented by the formula (1) is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

CRMFPNAPYL, (SEQ ID NO: 13) CCMTWNQMNL, (SEQ ID NO: 14) CCYTWNQMNL,(SEQ ID NO: 15) CALLPAVPSL, (SEQ ID NO: 16) CSLGEQQYSV (SEQ ID NO: 17)and CRVPGVAPTL, (SEQ ID NO: 18)or a pharmaceutically acceptable salt thereof;11. the compound according to any one of 1-8, wherein Fe is a grouprepresented by the formula (2), or a pharmaceutically acceptable saltthereof;12. the compound according to any one of 1-8 and 11, wherein X^(b) is adivalent peptide group consisting of 2 amino acid residues and Y^(b) isa single bond, or X^(b) and Y^(b) are each independently a divalentpeptide group consisting of 1 amino acid residue, or X^(b) is a singlebond and Y^(b) is a divalent peptide group consisting of 2 amino acidresidues, or X^(b) is a divalent peptide group consisting of 1 aminoacid residue and Y^(b) is a single bond, or X^(b) is a single bond andY^(b) is a divalent peptide group consisting of 1 amino acid residue, orX^(b) and Y^(b) are each a single bond, or a pharmaceutically acceptablesalt thereof;13. the compound according to any one of 1-8 and 11-12, wherein X^(b) isa single bond, and Y^(b) is a single bond, an alanine residue, a leucineresidue or a methionine residue, or a pharmaceutically acceptable saltthereof;14. the compound according to any one of 1-8 and 11-12, wherein X^(b) isa single bond or a divalent peptide group consisting of 1 amino acidresidue, and Y^(b) is a single bond, or a pharmaceutically acceptablesalt thereof;15. the compound according to any one of 1-8 and 11-14, wherein X^(b)and Y^(b) are each a single bond, or a pharmaceutically acceptable saltthereof;16. the compound according to any one of 1-8 and 11-15, wherein thecancer antigen peptide B is an MHC class I-restricted WT1 peptideconsisting of 7-15 amino acid residues, or a pharmaceutically acceptablesalt thereof;17. the compound according to any one of 1-8 and 11-16, wherein thecancer antigen peptide B is a peptide comprising any amino acid sequenceselected from the following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) ALLPAVPSL,(SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) and RVPGVAPTL, (SEQ ID NO: 7)ora peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 2, 3, 5, 6 and 7 but containingalteration of amino acid residue(s), and having a CTL inductionactivity, or a pharmaceutically acceptable salt thereof;18. the compound according to any one of 1-8 and 11-17, wherein thecancer antigen peptide B is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) CYTWNQMNL,(SEQ ID NO: 4) ALLPAVPSL, (SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) andRVPGVAPTL, (SEQ ID NO: 7)or a pharmaceutically acceptable salt thereof;19. the compound according to any one of 1-8 and 11-18, wherein thecompound represented by the formula (1) is a compound represented by theformula (3):

wherein the bond between C and C is a disulfide bond, or apharmaceutically acceptable salt thereof;20. the compound according to 13, wherein Y^(b) is an alanine residue,or a pharmaceutically acceptable salt thereof;21. the compound according to any one of 1-8 and 11-13 and 20, wherein,when the cancer antigen peptide B is an MHC class I-restricted WT1peptide containing one cysteine residue, the thioether group in thecancer antigen peptide B is bonded to the thioether group in the formula(16):

wherein X^(d) and Y^(d) are each independently a single bond or adivalent peptide group consisting of 1-4 amino acid residues, and atotal of the amino acid residue number for X^(d) and the amino acidresidue number for Y^(d) is an integer of 0-4, cancer antigen peptide Dis an MHC class II-restricted WT1 peptide consisting of 7-30 amino acidresidues, an amino group of an N-terminal amino acid of the cancerantigen peptide D binds to Y^(d) in the formula (16), and a carbonylgroup of a C-terminal amino acid of the cancer antigen peptide D bindsto a hydroxyl group in the formula (16), or to the thioether group ofthe cysteine residue of the cancer antigen peptide E, which is an MHCclass II-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue, or a pharmaceutically acceptable saltthereof;22. the compound according to 21, wherein the cancer antigen peptide Bis an MHC class I-restricted WT1 peptide consisting of 7-15 amino acidresidues, or a pharmaceutically acceptable salt thereof;23. the compound according to any one of 21-22, wherein the cancerantigen peptide B is a peptide consisting of any amino acid sequenceselected from the following amino acid sequences:

CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL, (SEQ ID NO: 4)or a pharmaceutically acceptable salt thereof;24. the compound according to any one of 21-23, wherein X^(d) is adivalent peptide group consisting of 2 amino acid residues and Y^(d) isa single bond, or X^(d) and Y^(d) are each independently a divalentpeptide group consisting of 1 amino acid residue, or X^(d) is a singlebond and Y^(d) is a divalent peptide group consisting of 2 amino acidresidues, or X^(d) is a divalent peptide group consisting of 1 aminoacid residue and Y^(d) is a single bond, or X^(d) is a single bond andY^(d) is a divalent peptide group consisting of 1 amino acid residue, orX^(d) and Y^(d) are each a single bond, or a pharmaceutically acceptablesalt thereof;25. the compound according to any one of 21-24, wherein X^(d) is asingle bond, Y^(d) is a single bond, an alanine residue, a leucineresidue or a methionine residue, or a pharmaceutically acceptable saltthereof;26. the compound according to any one of 21-24, wherein X^(d) is asingle bond or a divalent peptide group consisting of one amino acidresidue, and Y^(d) is a single bond, or a pharmaceutically acceptablesalt thereof;27. the compound according to any one of 21-26, wherein X^(d) and Y^(d)are each a single bond, or a pharmaceutically acceptable salt thereof;28. the compound according to any one of 21-27, wherein the cancerantigen peptide D is an MHC class II-restricted WT1 peptide consistingof 14-30 amino acid residues, or a pharmaceutically acceptable saltthereof;29. the compound according to any one of 21-28, wherein the cancerantigen peptide D is a peptide consisting of any amino acid sequenceselected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH,(SEQ ID NO: 23) CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24) andWAPVLDFAPPGASAYGSL, (SEQ ID NO: 244)or a pharmaceutically acceptable salt thereof;30. the compound according to any one of 1-8, 11-13 and 20-29, whereinthe compound represented by the formula (1) is a compound represented bythe formula (15):

wherein the bond between C and C is a disulfide bond, or apharmaceutically acceptable salt thereof;31. the compound according to any one of 21-23, wherein the cancerantigen peptide E is an MHC class II-restricted WT1 peptide consistingof 14-30 amino acid residues, or a pharmaceutically acceptable saltthereof;32. the compound according to any one of 21-23 and 31, wherein thecancer antigen peptide E is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH(SEQ ID NO: 23) and CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24)or a pharmaceutically acceptable salt thereof;33. the compound according to any one of 1-8, wherein R¹ is cancerantigen peptide C, or a pharmaceutically acceptable salt thereof;34. the compound according to any one of 1-8 and 33, wherein the cancerantigen peptide C is an MHC class I-restricted WT1 peptide consisting of7-15 amino acid residues, or a pharmaceutically acceptable salt thereof;35. the compound according to any one of 1-8 and 33-34, wherein thecancer antigen peptide C is a peptide comprising the following aminoacid sequence:

CMTWNQMNL, (SEQ ID NO: 3)ora peptide comprising an altered amino acid sequence, which is the aminoacid sequence of SEQ ID NO: 3 but containing alteration of amino acidresidue(s), and having a CTL induction activity, or a pharmaceuticallyacceptable salt thereof;36. the compound according to any one of 1-8 and 33-35, wherein thecancer antigen peptide C is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL, (SEQ ID NO: 4)or a pharmaceutically acceptable salt thereof;37. the compound according to any one of 1-8 and 33-36, wherein thecompound represented by the formula (1) is a compound represented by theformula (4):

wherein the bond between C and C is a disulfide bond, or a compoundrepresented by the formula (5):

wherein the bond between C and C is a disulfide bond,or a pharmaceutically acceptable salt thereof;38. the compound according to any one of 1-8 and 33, wherein, when thepeptide consisting of 1-4 amino acid residues containing one cysteineresidue is further bonded to the N-terminal of the cancer antigenpeptide C, the thioether group of the cysteine residue of the peptidebonded to the N-terminal of the cancer antigen peptide C is bonded tothe thioether group in the formula (16):

wherein X^(d) and Y^(d) are each independently a single bond or adivalent peptide group consisting of 1-4 amino acid residues, and atotal of the amino acid residue number for X^(d) and the amino acidresidue number for Y^(d) is an integer of 0-4, cancer antigen peptide Dis an MHC class II-restricted WT1 peptide consisting of 7-30 amino acidresidues, an amino group of an N-terminal amino acid of the cancerantigen peptide D binds to Y^(d) in the formula (16), and a carbonylgroup of a C-terminal amino acid of the cancer antigen peptide D bindsto a hydroxyl group in the formula (16), or to the thioether group ofthe cysteine residue of the cancer antigen peptide E, which is an MHCclass II-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue, or a pharmaceutically acceptable saltthereof;39. the compound according to 38, wherein the peptide consisting of 1-4amino acid residues containing one cysteine residue bonded to theN-terminal of the cancer antigen peptide C is a dipeptide consisting ofCA, or a pharmaceutically acceptable salt thereof;40. the compound according to any one of 38-39, wherein the cancerantigen peptide C is an MHC class I-restricted WT1 peptide consisting of7-15 amino acid residues, or a pharmaceutically acceptable salt thereof;41. the compound according to any one of 38-40, wherein the cancerantigen peptide C is a peptide consisting of any amino acid sequenceselected from the following amino acid sequences:

CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL, (SEQ ID NO: 4)or a pharmaceutically acceptable salt thereof;42. the compound according to any one of 38-41, wherein X^(d) is adivalent peptide group consisting of 2 amino acid residues and Y^(d) isa single bond, or X^(d) and Y^(d) are each independently a divalentpeptide group consisting of 1 amino acid residue, or X^(d) is a singlebond and Y^(d) is a divalent peptide group consisting of 2 amino acidresidues, or X^(d) is a divalent peptide group consisting of 1 aminoacid residue and Y^(d) is a single bond, or X^(d) is a single bond andY^(d) is a divalent peptide group consisting of 1 amino acid residue, orX^(d) and Y^(d) are each a single bond, or a pharmaceutically acceptablesalt thereof;43. the compound according to any one of 38-42, wherein X^(d) is asingle bond, and Y^(d) is a single bond, an alanine residue, a leucineresidue or a methionine residue, or a pharmaceutically acceptable saltthereof;44. the compound according to any one of 38-42, wherein X^(d) is asingle bond or a divalent peptide group consisting of 1 amino acidresidue, and Y^(d) is a single bond, or a pharmaceutically acceptablesalt thereof;45. the compound according to any one of 38-44, wherein X^(d) and Y^(d)are each a single bond, or a pharmaceutically acceptable salt thereof;46. the compound according to any one of 38-45, wherein the cancerantigen peptide D is an MHC class II-restricted WT1 peptide consistingof 14-30 amino acid residues, or a pharmaceutically acceptable saltthereof;47. the compound according to any one of 38-46, wherein the cancerantigen peptide D is a peptide consisting of any amino acid sequenceselected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH,(SEQ ID NO: 23) CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24) andWAPVLDFAPPGASAYGSL (SEQ ID NO: 244)or a pharmaceutically acceptable salt thereof;48. the compound according to any one of 38-47, wherein the compoundrepresented by the formula (1) is a compound represented by the formula(14):

wherein the bond between C and C is a disulfide bond,or a pharmaceutically acceptable salt thereof;49. the compound according to any one of 38-41 and 44, wherein thecancer antigen peptide E is a peptide consisting of any amino acidsequence selected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH(SEQ ID NO: 23) and CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24)or a pharmaceutically acceptable salt thereof;50. the compound according to any one of 38-41 and 49, wherein thecompound represented by the formula (1) is a compound represented by theformula (12):

wherein the bond between C and C is a disulfide bond, or apharmaceutically acceptable salt thereof;51. the compound according to any one of 1-8 and 33, wherein the cancerantigen peptide C is an MHC class II-restricted WT1 peptide consistingof 14-30 amino acid residues, or a pharmaceutically acceptable saltthereof;52. the compound according to any one of 1-8, 33 and 51, wherein thecancer antigen peptide C is a peptide comprising any amino acid sequenceselected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11)PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20) PGCNKRYFKLSHLQMHSRKH,(SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 10)CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH (SEQ ID NO: 23)and CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24)ora peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 10-11 and 19-24 but containingalteration of amino acid residue(s), and having a helper T cellinduction activity, or a pharmaceutically acceptable salt thereof;53. the compound according to any one of 1-8, 33 and 51-52, wherein thecancer antigen peptide C is a peptide consisting of an amino acidsequence selected from the following amino acid sequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH(SEQ ID NO: 23) and CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24)or a pharmaceutically acceptable salt thereof;54. the compound according to any one of 1-8, 33 and 51-53, wherein thecompound represented by the formula (1) is a compound represented by theformula (6):

wherein the bond between C and C is a disulfide bond,a compound represented by the formula (7):

wherein the bond between C and C is a disulfide bond, a compoundrepresented by the formula (8):

wherein the bond between C and C is a disulfide bond, or a compoundrepresented by the formula (9):

wherein the bond between C and C is a disulfide bond,or a pharmaceutically acceptable salt thereof;55. an altered form of an MHC class II-restricted WT1 peptide consistingof 7-30 amino acid residues;56. the altered form according to 55, which is the following amino acidsequence:

CWAPVLDFAPPGASAYGSL (SEQ ID NO: 242) or WAPVLDFAPPGASAYGSLC;(SEQ ID NO: 243)57. a compound represented by the formula (10):

wherein the bond between C and C is a disulfide bond,or a pharmaceutically acceptable salt thereof;58. a composition comprising a compound selected from the groupconsisting of a compound represented by the formula (3):

wherein the bond between C and C is a disulfide bond, a compoundrepresented by the formula (4):

wherein the bond between C and C is a disulfide bond, and a compoundrepresented by the formula (5):

wherein the bond between C and C is a disulfide bond, and a peptideconsisting of an amino acid sequence selected from the group consistingof the following amino acid sequences:

CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH, (SEQ ID NO: 23)CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24) WAPVLDFAPPGASAYGSL,(SEQ ID NO: 244) CWAPVLDFAPPGASAYGSL (SEQ ID NO: 242) andWAPVLDFAPPGASAYGSLC; (SEQ ID NO: 243)59. a pharmaceutical composition comprising the compound according toany one of 1-54 and 57, or a pharmaceutically acceptable salt thereof,or the composition according to 58 and a pharmaceutically acceptablecarrier;60. the pharmaceutical composition according to 59, which is used as acancer vaccine;61. use of the compound according to any one of 1-54 and 57, or apharmaceutically acceptable salt thereof, or the composition accordingto 58 for the production of a cancer vaccine;62. a method of treating or preventing cancer, comprising administeringa therapeutically or prophylactically effective amount of the compoundaccording to any one of 1-54 and 57 or a pharmaceutically acceptablesalt thereof or the composition according to 58 to a WT1 positive cancerpatient in need thereof;63. a method of obtaining two different MHC class I-restricted epitopes,or an MHC class I-restricted epitope and an MHC class II-restrictedepitope, comprising reacting the compound according to any one of 1-54and 57 or a pharmaceutically acceptable salt thereof with ERAP1; and64. a method of synthesizing a compound, comprising the following steps:(1) a step of synthesizing, by using Fmoc-C(Mmt)A-SBn and cancer antigenpeptide C, a peptide wherein a carbonyl group of the C-terminal aminoacid of C(Mmt)A and the N-terminal amino group of the cancer antigenpeptide C are bonded, wherein the antigen peptide C is an MHC classI-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue or an MHC class II-restricted WT1peptide consisting of 7-30 amino acid residues containing one cysteineresidue,(2) a step of synthesizing, by using the peptide obtained in theaforementioned step (1) and cancer antigen peptide A wherein onecysteine residue protected by Npys group is bonded to the N-terminal, apeptide wherein a thioether group of the cysteine residue of the cancerantigen peptide C in the peptide obtained in the aforementioned step (1)and a thioether group of the cysteine residue bonded to the N-terminalof cancer antigen peptide A are bonded, wherein the cancer antigenpeptide A is an MHC class I-restricted WT1 peptide consisting of 7-30amino acid residues, and(3) a step of synthesizing, by using the peptide obtained in theaforementioned step (2) and cancer antigen peptide D containing acysteine residue protected by Spy group, a peptide wherein a thioethergroup of the cysteine residue bonded to the N-terminal of the cancerantigen peptide A in the peptide obtained in the aforementioned step(2), and a thioether group of the cysteine residue of the cancer antigenpeptide D are bonded, wherein the cancer antigen peptide D is an MHCclass II-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue bonded to the N terminal or an MHC classII-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue,

3. Third Embodiment

1. A compound represented by the formula (1)

wherein X^(a) and Y^(a) are each a single bond, cancer antigen peptide Ais a peptide consisting of any amino acid sequence selected from thefollowing amino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) ALLPAVPSL, (SEQ ID NO: 5) SLGEQQYSV(SEQ ID NO: 6) and RVPGVAPTL, (SEQ ID NO: 7)an amino group of an N-terminal amino acid of the cancer antigen peptideA binds to Y^(a) in the formula (1), and a carbonyl group of aC-terminal amino acid of the cancer antigen peptide A binds to ahydroxyl group in the formula (1),R¹ is a cancer antigen peptide C,the cancer antigen peptide C has a sequence different from that of thecancer antigen peptide A, which is a peptide consisting of any aminoacid sequence selected from the following amino acid sequences:

CMTWNQMNL (SEQ ID NO: 3) and

CYTWNQMNL, (SEQ ID NO: 4)residue of the cancer antigen peptide C is bonded to the thioether groupin the formula (1),or a pharmaceutically acceptable salt thereof;2. the compound according to 1, wherein the compound represented by theformula (1) is a compound represented by the formula (4):

wherein the bond between C and C is a disulfide bond, or apharmaceutically acceptable salt thereof;3. the compound according to 1, wherein the compound represented by theformula (1) is a compound represented by the formula (5):

wherein the bond between C and C is a disulfide bond,or a pharmaceutically acceptable salt thereof;4. a pharmaceutical composition comprising the compound according to anyone of 1-3, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier;5. the pharmaceutical composition according to 4, comprising at leastone peptide consisting of an amino acid sequence selected from the groupconsisting of the following amino acid sequences:

CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH, (SEQ ID NO: 23)CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24) WAPVLDFAPPGASAYGSL,(SEQ ID NO: 244) CWAPVLDFAPPGASAYGSL (SEQ ID NO: 242) andWAPVLDFAPPGASAYGSLC; (SEQ ID NO: 243)and6. a composition comprising a compound selected from the groupconsisting of a compound represented by the formula (4):

wherein the bond between C and C is a disulfide bond, and a compoundrepresented by the formula (5):

wherein the bond between C and C is a disulfide bond, and at least onepeptide consisting of an amino acid sequence selected from the groupconsisting of the following amino acid sequences:

CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH, (SEQ ID NO: 23)CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24) WAPVLDFAPPGASAYGSL,(SEQ ID NO: 244) CWAPVLDFAPPGASAYGSL (SEQ ID NO: 242) andWAPVLDFAPPGASAYGSLC. (SEQ ID NO: 243)

Effect of the Invention

According to the present invention, the aforementioned compoundrepresented by the formula (1) useful as a cancer immunotherapeuticagent (hereinafter sometimes to be referred to as the compound of thepresent invention) can be provided. According to the compound of thepresent invention, cancer vaccines and cancer immunotherapeutic agentsthat efficiently induce CTL in vivo and in vitro can be provided. To bespecific, according to the compound of the present invention, it is nowpossible to produce two MHC class I-restricted WT1 peptides havingdifferent sequences or two MHC class I-restricted WT1 epitopes havingdifferent sequences, an MHC class I-restricted WT1 peptide and an MHCclass II-restricted WT1 peptide, an MHC class I-restricted WT1 epitopeand an MHC class II-restricted WT1 epitope, two MHC class I-restrictedWT1 peptide and MHC class II-restricted WT1 peptide having differentsequences, or two MHC class I-restricted WT1 epitope and MHC classII-restricted WT1 epitope having different sequences in vivo and invitro and efficiently induce CTL.

As for the HLA subtypes of two MHC class I-restricted WT1 peptideshaving different sequence, the compound of the present invention(conjugate) obtained by combining A02 type (A-0201, A0206 and the like)peptide and A24 type (A-2402 and the like) peptide is particularlypreferable. In Europeans and Americans (Caucasian), the population ofHLA-A0201 subtype or HLA-A0206 subtype is the highest and about 470,then HLA-A2402 subtype is about 13%, and the total of these subtypesoccupies about 56%, excluding duplicates (i.e., duplicate calculation ofhumans having both subtypes) (Human Immunol. 62:1009; 2001). In Japanesepeople and the like, the population of HLA-A2402 is the highest andabout 60%, then HLA-A0201 or HLA-A0206 is about 39%, and the total ofthese subtypes occupies about 81%, excluding duplicates (i.e., duplicatecalculation of humans having both subtypes)(www.bmdc.irc.or.jp/GF-A.htm). Therefore, the advantages of the compoundof the present invention are, specifically, that a larger population iscovered by a single compound of the present invention, and selection ofthe HLA subtype of the patients before administration is not alwaysessential and the like. In view of such advantages of the compound ofthe present invention, a compound represented by the formula (3), theformula (4) or the formula (5) is preferable, and a compound representedby the formula (5) is more preferable.

Moreover, according to the compound of the present invention, an activeingredient of a cancer vaccine superior in physicochemical propertiesand stability, and easily produced and easily controlled can beprovided. As a result, formulation of cancer vaccine has beenfacilitated.

Specifically, examples of the physicochemical properties includesolubility, viscosity of solution, easiness of purification resultingtherefrom, easy handling after freeze-drying, easiness of purificationresulting therefrom and the like. The stability includes stability aftersalt substitution, hygroscopicity, thermal stability, stability afteremulsion formation and the like. The pharmacological activity includesefficacy as cancer vaccine, difference caused by API (ActivePharmaceutical Ingredient), interaction with additive in preparation andthe like. Of these, the difference caused by API is a difference as acancer vaccine due to API. Specifically, in two APIs having vastlydifferent solubilities, API with smaller solubility is prone toprecipitate, and it is easily expected that a sterilization treatment byfiltration with a membrane filter, which is an essential requirement forpharmaceutical products, cannot be performed. Even if a sterilizationtreatment by filtration of API with small solubility is barely possible,it is considered that the amount of API contained in the filtratemarkedly decreases and CTL induction ability essential for a cancervaccine markedly decreases. Therefore, a demerit of markedly decreasedproduction efficiency of API with small solubility is easilypredictable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Figure showing the test results of Experimental Example 1 asto the time-dependent change of N-terminal amino acid trimming by ERAP1of each peptide of SEQ ID NOs: 13, 16, 17 and 18 synthesized in Examples2-5.

FIG. 2 is a Figure showing the test results of Experimental Example 2 asto the in vivo CTL induction ability of a compound represented by theformula (5) synthesized in Example 1, by IFNγ ELISPOT assay usingHLA-A0201 transgenic mouse.

FIG. 3 is a Figure showing the test results of Experimental Example 2 asto the in vivo CTL induction ability of a compound represented by theformula (5) synthesized in Example 1, by IFNγ ELISPOT assay usingHLA-A2402 transgenic mouse.

FIG. 4 is a Figure showing the test results of Experimental Example 4 asto the in vivo CTL induction ability of a compound represented by theformula (3) synthesized in Example 6, by IFNγ ELISPOT assay usingHLA-A0201 transgenic mouse.

FIG. 5 is a Figure showing the test results of Experimental Example 5 asto the ability of a compound represented by the formula (6) synthesizedin Example 7 to induce cells reactive with peptide shown by SEQ ID NO:24 in vivo, by IFNγ ELISPOT assay using HLA-A0201 transgenic mouse inthe pulsed or non-pulsed state with peptide of SEQ ID NO: 24.

FIG. 6 is a Figure showing the test results of Experimental Example 5 asto the ability of a compound represented by the formula (6) synthesizedin Example 7 to induce cells reactive with peptide shown by SEQ ID NO:24 in vivo, by IFNγ ELISPOT assay using HLA-A0201 transgenic mouse inthe pulsed or non-pulsed state with peptide of SEQ ID NO: 24.

FIG. 7 is a Figure showing the test results of Experimental Example 6 asto the in vivo CTL induction ability of a compound represented by theformula (8) synthesized in Example 9 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 2, by IFNγ ELISPOT assay using HLA-A0201transgenic mouse.

FIG. 8 is a Figure showing the test results of Experimental Example 6 asto the ability of a compound represented by the formula (8) synthesizedin Example 9 to induce cells reactive with peptide shown by SEQ ID NO:22 in vivo, by IFNγ ELISPOT assay using HLA-A0201 transgenic mouse inthe pulsed or non-pulsed state with peptide of SEQ ID NO: 22.

FIG. 9 is a Figure showing the test results of Experimental Example 8 asto the in vivo CTL induction ability of a compound represented by theformula (7) synthesized in Example 8 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 2, by IFNγ ELISPOT assay using HLA-A0201transgenic mouse.

FIG. 10 is a Figure showing the test results of Experimental Example 8as to the ability of a compound represented by the formula (7)synthesized in Example 8 to induce cells reactive with peptide shown bySEQ ID NO: 23 in vivo, by IFNγ ELISPOT assay using HLA-A0201 transgenicmouse in the pulsed or non-pulsed state with peptide of SEQ ID NO: 23.

FIG. 11 is a Figure showing the test results of Experimental Example 9as to the in vivo CTL induction ability of a compound represented by theformula (9) synthesized in Example 10 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 5, by IFNγ ELISPOT assay using HLA-A0201transgenic mouse.

FIG. 12 is a Figure showing the test results of Experimental Example 9as to the ability of a compound represented by the formula (9)synthesized in Example 10 to induce cells reactive with peptide shown bySEQ ID NO: 24 in vivo, by IFNγ ELISPOT assay using HLA-A0201 transgenicmouse in the pulsed or non-pulsed state with peptide of SEQ ID NO: 24.

FIG. 13 is a Figure showing the test results of Comparative Example 1 asto the in vivo CTL induction ability of peptides shown by SEQ ID NO: 238and 239 synthesized in Reference Examples 8 and 9 in the pulsed ornon-pulsed state with peptide of SEQ ID NO: 2, by IFNγ ELISPOT assayusing HLA-A0201 transgenic mouse.

FIG. 14 is a Figure showing the test results of Comparative Example 1 asto the in vivo CTL induction ability of peptides shown by SEQ ID NO: 238and 239 synthesized in Reference Examples 8 and 9 in the pulsed ornon-pulsed state with peptide of SEQ ID NO: 4, by IFNγ ELISPOT assayusing HLA-A2402 transgenic mouse.

FIG. 15 is a Figure showing the test results of Comparative Example 2 asto the in vivo CTL induction ability of peptides shown by SEQ ID NO: 240and 241 synthesized in Reference Examples 10 and 11 in the pulsed ornon-pulsed state with peptide of SEQ ID NO: 2, by IFNγ ELISPOT assayusing HLA-A0201 transgenic mouse.

FIG. 16 is a Figure showing the test results of Comparative Example 2 asto the in vivo CTL induction ability of peptides shown by SEQ ID NO: 240and 241 synthesized in Reference Examples 10 and 11 in the pulsed ornon-pulsed state with peptide of SEQ ID NO: 4, by IFNγ ELISPOT assayusing HLA-A2402 transgenic mouse.

FIG. 17 is a Figure showing the test results of Experimental Example 11as to the in vivo CTL induction ability of a compound represented by theformula (10) synthesized in Example 13 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 2, by IFNγ ELISPOT assay using HLA-A0201transgenic mouse.

FIG. 18 is a Figure showing the test results of Comparative Example 3 asto the in vivo CTL induction ability of a compound represented by theformula (11) synthesized in Reference Example 12 in the pulsed ornon-pulsed state with peptide of SEQ ID NO: 2, by IFNγ ELISPOT assayusing HLA-A0201 transgenic mouse.

FIG. 19 is a Figure showing the test results of Experimental Example 12as to the in vivo CTL induction ability of a compound represented by theformula (12) synthesized in Example 14 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 2, by IFNγ ELISPOT assay using HLA-A0201transgenic mouse.

FIG. 20 is a Figure showing the test results of Experimental Example 12as to the in vivo CTL induction ability of a compound represented by theformula (12) synthesized in Example 14 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 4, by IFNγ ELISPOT assay using HLA-A2402transgenic mouse.

FIG. 21 is a Figure showing the test results of Experimental Example 13as to the in vivo CTL induction ability of a compound represented by theformula (14) synthesized in Example 15 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 2, by IFNγ ELISPOT assay using HLA-A0201transgenic mouse.

FIG. 22 is a Figure showing the test results of Experimental Example 13as to the in vivo CTL induction ability of a compound represented by theformula (14) synthesized in Example 15 in the pulsed or non-pulsed statewith peptide of SEQ ID NO: 4, by IFNγ ELISPOT assay using HLA-A2402transgenic mouse.

FIG. 23 is a Figure showing the test results of Experimental Example 14as to the in vivo CTL induction ability of a cocktail vaccine of acompound represented by the formula (5) synthesized in Example 1 and thepeptide shown by SEQ ID NO: 22 synthesized in Reference Example 1 in thepulsed or non-pulsed state with peptide of SEQ ID NO: 2, by IFNγ ELISPOTassay using HLA-A0201 transgenic mouse.

FIG. 24 is a Figure showing the test results of Experimental Example 15as to the in vivo CTL induction ability of a cocktail vaccine of acompound represented by the formula (5) synthesized in Example 1 and thepeptide shown by SEQ ID NO: 244 synthesized in Reference Example 13 inthe pulsed or non-pulsed state with peptide of SEQ ID NO: 2, by IFNγELISPOT assay using HLA-A0201 transgenic mouse.

FIG. 25 is a Figure showing the test results of Experimental Example 16as to the in vivo CTL induction ability of a cocktail vaccine of acompound represented by the formula (5) synthesized in Example 1 and thepeptide shown by SEQ ID NO: 24 synthesized in Reference Example 2 in thepulsed or non-pulsed state with peptide of SEQ ID NO: 2, by IFNγ ELISPOTassay using HLA-A0201 transgenic mouse.

FIG. 26 is a Figure showing the test results of Experimental Example 17as to the in vivo CTL induction ability of a cocktail vaccine of acompound represented by the formula (5) synthesized in Example 1 and thepeptide shown by SEQ ID NO: 242 synthesized in Example 11 in the pulsedor non-pulsed state with peptide of SEQ ID NO: 2, by IFNγ ELISPOT assayusing HLA-A0201 transgenic mouse.

FIG. 27 is a Figure showing the test results of Experimental Example 18as to the in vivo CTL induction ability of a cocktail vaccine of acompound represented by the formula (5) synthesized in Example 1 and thepeptide shown by SEQ ID NO: 243 synthesized in Example 11 in the pulsedor non-pulsed state with peptide of SEQ ID NO: 2, by IFNγ ELISPOT assayusing HLA-A0201 transgenic mouse.

DESCRIPTION OF EMBODIMENTS

The embodiment of the present invention is explained in detail in thefollowing.

The “amino acid residue” in the present invention means a regioncorresponding to one unit of amino acid constituting a peptide orprotein in a peptide or protein molecule. Examples of the “amino acidresidue” include natural or non-natural α-amino acid residue, (3-aminoacid residue, γ-amino acid residue or δ-amino acid residue. Specificexamples thereof include natural α-amino acid residue, ornithineresidue, homoserine residue, homocysteine residue, (3-alanine,γ-aminobutanoic acid or δ-aminopentanoic acid and the like. When the“amino acid residue” can be an optically active substance, it may be anyof an L-form and a D-form, and an L-form is preferable.

When the “amino acid residue” in the present invention is shown inabbreviation, the following abbreviations are used.

Ala or A: alanine residue

Arg or R: arginine residue

Asn or N: asparagine residue

Asp or D: aspartic acid residue

Cys or C: cysteine residue

Gln or Q: glutamine residue

Glu or E: glutamic acid residue

Gly or G: glycine residue

His or H: histidine residue

Ile or I: isoleucine residue

Leu or L: leucine residue

Lys or K: lysine residue

Met or M: methionine residue

Phe or F: phenylalanine residue

Pro or P: proline residue

Ser or S: serine residue

Thr or T: threonine residue

Trp or W: tryptophan residue

Tyr or Y: tyrosine residue

Val or V: valine residue

Abu: 2-aminobutyric acid residue (to be also referred to asα-aminobutyric acid residue)

Orn: ornithine residue

Cit: citrulline residue

The amino acid sequence of the “peptide” in the present invention isdescribed according to the conventional method, wherein the amino acidresidue of the N-terminal amino acid is positioned on the left side, andthe amino acid residue of the C-terminal amino acid is positioned on theright side. In the “peptide”, unless particularly indicated, the aminogroup of the amino acid residue of the N-terminal amino acid is bondedto hydrogen atom, and the carbonyl group of the amino acid residue ofthe C-terminal amino acid is bonded to hydroxyl group. The divalentgroup of peptide means a group bonding via the amino group of the aminoacid residue of the N-terminal amino acid and the carbonyl group of theamino acid residue of the C-terminal amino acid.

In the compound of the present invention, for example, in the compoundsrepresented by the formulae (3)-(9) and as regards peptide, which is apartial structure thereof, unless particularly indicated, the aminogroup of the amino acid residue of the N-terminal amino acid is bondedto hydrogen atom, and the carbonyl group of the amino acid residue ofthe C-terminal amino acid is bonded to hydroxyl group.

“X^(a)” and “Y^(a)” in the present invention mean, independently, asingle bond or a divalent group of peptides consisting of 1-4 amino acidresidues. The sum of the amino acid residue number of X^(a) and that ofY^(a) is an integer of 0-4. For example, an integer of said sum being 0means that X^(a) and Y^(a) are each a single bond. When the sum is aninteger of 4, examples thereof include X^(a) and Y^(a) independentlybeing divalent groups of peptide consisting of 2 amino acid residues,X^(a) being a divalent group of peptide consisting of 3 amino acidresidues and Y^(a) being a divalent group of peptide consisting of 1amino acid residue, X^(a) being a divalent group of peptide consistingof 4 amino acid residues and Y^(a) being a single bond and the like.

The integer of said sum is preferably 0-2, more preferably 0-1, mostpreferably 0. That is, X^(a) and Y^(a) are most preferably single bonds.

When the sum is an integer of 2, examples thereof include X^(a) being adivalent group of peptide consisting of 2 amino acid residues and Y^(a)being a single bond, X^(a) and Y^(a) independently being divalent groupsof peptide consisting of 1 amino acid residue, or X^(a) being a singlebond and Y^(a) being a divalent group of peptide consisting of 2 aminoacid residues.

When the sum is an integer of 1, examples thereof include X^(a) being adivalent group of peptide consisting of 1 amino acid residue and Y^(a)being a single bond, and X^(a) being a single bond and Y^(a) being adivalent group of peptide consisting of 1 amino acid residue. Of these,preferred is X^(a) being a single bond and Y^(a) being an alanineresidue, leucine residues or methionine residue.

The “cancer antigen peptide A” in the present invention is an MHC classI-restricted WT1 peptide consisting of 7-30 amino acid residues. Incancer antigen peptide A in the formula (1), the amino group of theN-terminal amino acid is bonded to Y^(a) in the formula (1) and thecarbonyl group of the C-terminal amino acid is bonded to the hydroxylgroup in the formula (1).

The “MHC class I-restricted” in the present invention means the propertyto induce CTL by binding to an MHC class I molecule which is class I ofthe major histocompatibility complex (MHC).

MHC in human is called human leukocyte-type antigen (HLA). HLAcorresponding to the MHC class I-molecule is classified into subtypes ofHLA-A, B, Cw, F and G and the like. Preferable examples of the “MHCclass I-restricted” include HLA-A-restricted, HLA-B-restricted andHLA-Cw-restricted.

Polymorphism (allele) of each subtype of HLA is known. Examples of thepolymorphism of HLA-A include not less than 27 kinds such as HLA-A1,HLA-A0201, HLA-A24 and the like, examples of the polymorphism of HLA-Binclude not less than 59 kinds such as HLA-B7, HLA-B40, HLA-B4403 andthe like, and examples of the polymorphism of HLA-Cw include not lessthan 10 kinds such as HLA-Cw0301, HLA-Cw0401, HLA-Cw0602 and the like.Among these polymorphisms, HLA-A0201 and HLA-A24 are preferable.

The “WT1 peptide” in the present invention is a partial peptideconsisting of continuous 7-30 amino acid residues in the amino acidsequence of human WT1 described in SEQ ID NO: 1.

Therefore, the “MHC class I-restricted WT1 peptide” in the presentinvention is a peptide that binds to an MHC class I antigen in vitroand/or in vivo and is presented as a complex, and induces CTL as aresult of recognition of the complex by precursor T cells. The number ofthe amino acid residues of the “MHC class I-restricted WT1 peptide” is7-30, preferably 7-15, more preferably 8-12, further preferably 8-11,most preferably 8 or 9.

The “MHC class I-restricted WT1 peptide” consisting of 7-12 orpreferably 9 amino acid residues is also called “an MHC classI-restricted WT1 epitope”. The “MHC class I-restricted WT1 epitope” inthe present invention means a peptide per se that binds to an MHC classI antigen and is presented as a complex. That is, “MHC classI-restricted WT1 peptide” produces “MHC class I-restricted WT1 epitope”in vitro and/or in vivo, by intracellular decomposition of the compoundof the present invention by proteosome and/or protease such asGamma-Interferon-inducible Lysosomal Thiol Reductase (GILT, GLT) and thelike (proteolysis, reductive cleavage of disulfide bond), and/orcleavage into the optimal residue number (also called trimming) byEndoplasmic reticulum aminopeptidase 1 (ERAP1, ER-aminopeptidase 1). Inthis production, a production process wherein the C-terminal amino acidof the “MHC class I-restricted WT1 epitope” first results from thedegradation by proteosome and/or protease, after which N-terminal aminoacid of the “MHC class I-restricted WT1 epitope” results from trimming(cleavage) by ERAP1 is mainly considered. In this production, however, aprocess other than this production process is also possible. At present,ERAP1 is also referred to as ERAAP (ER aminopeptidase associated withantigen presentation), and used to be also called A-LAP, PILS-AP orARTS-1.

Therefore, the “MHC class I-restricted WT1 peptide” is preferably apeptide consisting of 7-30 amino acid residues produced by adding 1-23amino acid residues to the carbonyl group of the C-terminal amino acidof the “MHC class I-restricted WT1 epitope” consisting of 7-12 aminoacid residues.

Examples of the “MHC class I-restricted WT1 peptide” include peptidesdescribed in Tables 1-44. In each Table, the “position” means a positionin the amino acid sequence of human WT1 described in SEQ ID NO: 1.

TABLE 1 amino acid sequence HLA position sequence No. subtype  2-10GSDVRDLNA 26 A1  3-11 SDVRDLNAL 27 B40, B60, B61, B3701, B4403,Cw0301, Cw0602  4-12 DVRDLNALL 28 A24, A68.1, A3302, B7, B8,B3501, B3701, Cw0401, Cw0602  6-14 RDLNALLPA 29 B40, B61, B3701  7-15DLNALLPAV 30 A0201, B62, B5201 10-18 ALLPAVPSL  5 A0201, A0205,A24, A3, B14, B7, B8, B3801, B3901, B3902, Cw0301, Cw0401, Cw0602

TABLE 2 amino acid sequence HLA position sequence No. subtype 17-25SLGGGGGCA 31 B62 18-26 LGGGGGCAL 32 B60, B7, B3801, B5101, B5102 20-28GGGGCALPV 33 B61, B5101, B5102, B5201

TABLE 3 amino acid sequence HLA position sequence No. subtype 23-31GCALPVSGA 34 B40, B61 24-32 CALPVSGAA 35 B40, B5102, Cw0301 26-34LPVSGAAQW 36 B40, B3501, B5801 29-37 SGAAQWAPV 37 B5101, B5102,B5201, B61 30-38 GAAQWAPVL 38 B40, B60, B7, B8, B3902, B5101, B5102,Cw0301, Cw0602

TABLE 4 amino acid sequence HLA position sequence No. subtype 32-40AQWAPVLDF 39 A3, A3101, B62, B2702, B2705, B3902, B5201 33-41 QWAPVLDFA40 Cw0702 37-45 VLDFAPPGA 41 A1, A0201 38-46 LDFAPPGAS 42 B40, B370139-47 DFAPPGASA 43 Cw0401 40-48 FAPPGASAY 44 A1, B62, B3501, B4403,B5801, Cw0702

TABLE 5 amino acid sequence HLA position sequence No. subtype 47-55AYGSLGGPA 45 A24

TABLE 6 amino acid sequence HLA position sequence No. subtype 63-71PPPPPPHSF 46 Cw0401 64-72 PPPPPHSFI 47 B5101, B5102, B5201 65-73PPPPHSFIK 48 A1101 70-78 SFIKQEPSW 49 Cw0401

TABLE 7 amino acid sequence HLA position sequence No. subtype 73-81KQEPSWGGA 50 A1, A0205 80-88 GAEPHEEQC 51 A1

TABLE 8 amino acid sequence HLA position sequence No. subtype 81-89AEPHEEQCL 52 A0205, B40, B60, B61, B3701, B4403 82-90 EPHEEQCLS 53B3501, B5101 83-91 PHEEQCLSA 54 B3801 84-92 HEEQCLSAF 55 B40, B3701,B4403, Cw0702 85-93 EEQCLSAFT 56 B40, B60, B61, B3701, B4403 86-94EQCLSAFTV 57 A0201, B62, B5201 88-96 CLSAFTVHF 58 A3, B62

TABLE 9 amino   acid sequence HLA position sequence No. subtype  92-100FTVHFSGQF 59 B62, B5801, Cw0301  93-101 TVHFSGQFT 60 A0201, A0205 96-104 FSGQFTGTA 61 B5801, B4403  98-106 GQFTGTAGA 62 A0205, B40,B62, B2702, B5201  99-107 QFTGTAGAC 63 Cw0401 100-108 FTGTAGACR 64A68.1, A1101, A3101, A3302

TABLE 10 amino acid sequence HLA position sequence No. subtype 101-109TGTAGACRY 65 B62, B4403, Cw0702 104-112 AGACRYGPF 66 B4403, B5201107-115 CRYGPFGPP 67 B2702 110-118 GPFGPPPPS 68 B5101, B5102

TABLE 11 amino acid sequence HLA position sequence No. subtype 118-126SQASSGQAR 69 A68.1, A1101, A3101, A3302 119-127 QASSGQARM 70B3501, B5101, B5102 120-128 ASSGQARMF 71 B3501, B3801, B4403, B5801

TABLE 12 amino acid sequence HLA position sequence No. subtype 123-131GQARMFPNA 72 B62 125-133 ARMFPNAPY 73 B14, B2702, B2705, Cw0702 126-134RMFPNAPYL 2 A0201, A0205, A24, A3, B14, B7, B2702, B2705, B3901,B3902, Cw0301 128-136 FPNAPYLPS 74 B5101 130-138 NAPYLPSCL 75A24, B60, B7, B8, B3902, B5101,B5102, Cw0301, Cw0602,Cw0702

TABLE 13 amino acid sequence HLA position sequence No. subtype 136-144SCLESQPAI 76 B8, B3901, B5102, Cw0301 137-145 CLESQPAIR 77A1, A3, A68.1, A1101, A3101, A3302 138-146 LESQPAIRN 78 B60, B61 139-147ESQPAIRNQ 79 A3302

TABLE 14 amino acid sequence HLA position sequence No. subtype 141-149QPAIRNQGY 80 B8, B3501, B4403, Cw0401, Cw0702 143-151 AIRNQGYST 81 B7144-152 IRNQGYSTV 82 B14, B2702, B2705, B3901 146-154 NQGYSTVTF 83B62, B2702, B3902, B5201

TABLE 15 amino acid sequence HLA position sequence No. subtype 152-160VTFDGTPSY 84 A1, A3, B62, B3501, B4403, B5801, Cw0702

TABLE 16 amino acid sequence HLA position sequence No. subtype 161-169GHTPSHHAA 85 B3801 163-171 TPSHHAAQF 86 B3501, B3801, Cw0401, Cw0702165-173 SHHAAQFPN 87 B3801 168-176 AAQFPNHSF 88 B5801 169-177 AQFPNHSFK89 A3, A68.1, A1101, A3101, B2705

TABLE 17 amino acid sequence HLA position sequence No. subtype 174-182HSFKHEDPM 90 B14, B3501, B5801 177-185 KHEDPMGQQ 91 B3801 179-187EDPMGQQGS 92 B3701 180-188 DPMGQQGSL 93 A24, B14, B60, B7, B8, B3501,B3801, B3901, B3902, B5101, B5102, Cw0301, Cw0401, Cw0602

TABLE 18 amino acid sequence HLA position sequence No. subtype 185-193QGSLGEQQY 94 B4403, Cw0702 187-195 SLGEQQYSV  6 A0201, A0205, A3, B62

TABLE 19 amino acid sequence HLA position sequence No. subtype 191-199QQYSVPPPV 95 A0201, A0205, B61, B62, B2702, B2705, B5201 192-200QYSVPPPVY 96 A24, Cw0401, Cw0702 194-202 SVPPPVYGC 97 A0205, A3

TABLE 20 amino acid sequence HLA position sequence No. subtype 202-210CHTPTDSCT  98 B3801 204-212 TPTDSCTGS  99 B5101 206-214 TDSCTGSQA 100B40, B61, B3701 207-215 DSCTGSQAL 101 A24, A3302, B60, B7, B8,B3501, B3901, B3902, Cw0602 208-216 SCTGSQALL 102 B60, B7, B8,B3701, B3801, B3901, B3902 209-217 CTGSQALLL 103 B60, B7, B3701, B3902210-218 TGSQALLLR 104 A3302

TABLE 21 amino acid sequence HLA position sequence No. subtype 211-219GSQALLLRT 105 B5801 213-221 QALLLRTPY 106 A1, B3501, B4403, B5801,Cw0602, Cw0702 217-225 LRTPYSSDN 107 B2702 218-226 RTPYSSDNL 108A24, B60, B7, B3902, B5801 219-227 TPYSSDNLY 109 B3501, B5101,B5102, Cw0401, Cw0702

TABLE 22 amino acid sequence HLA position sequence No. subtype 221-229YSSDNLYQM 110 B60, B3501 222-230 SSDNLYQMT 111 A1, B5801 223-231SDNLYQMTS 112 B3701 225-233 NLYQMTSQL 113 A0201, A0205, A24, B14, B7,B8, B3801, B3901, B3902, Cw0301, Cw0602 227-235 YQMTSQLEC 114A0201, A0205, B62 228-236 QMTSQLECM 115 A0201 230-238 TSQLECMTW 116B5801

TABLE 23 amino acid sequence HLA position sequence No. subtype 232-240QLECMTWNQ 117 A1 233-241 LECMTWNQM 118 B40, B60, B61, B3701, B4403235-243 CMTWNQMNL   3 A0201, A0205, A24, A3, B7 239-247 NQMNLGATL 119A0201, A0205, A24, B14, B60, B62, B7, B2702, B2705, B3901, B3902,B5201, Cw0301, Cw0602 240-248 QMNLGATLK 120 A24, A3, A1101, A3101

TABLE 24 amino acid sequence HLA position sequence No. subtype 242-250NLGATLKGV 121 A0201, A0205, B62, Cw0602 243-251 LGATLKGVA 122 B5201244-252 GATLKGVAA 123 B61, B8 250-258 VAAGSSSSV 124 B61, B5101, B5102

TABLE 25 amino acid sequence HLA position sequence No. subtype 251-259AAGSSSSVK 125 A68.1, A1101 252-260 AGSSSSVKW 126 B5801 260-268 WTEGQSNHS127 A1

TABLE 26 amino acid sequence HLA position sequence No. subtype 261-269TEGQSNHST 128 B40, B60, B61, B4403 263-271 GQSNHSTGY 129 A3, B62,B2702, Cw0702 269-277 TGYESDNHT 130 B5102, B5201 270-278 GYESDNHTT 131A24

TABLE 27 amino acid sequence HLA position sequence No. subtype 272-280ESDNHTTPI 132 A1, A3302, B5101 273-281 SDNHTTPIL 133 B40, B60,B3701, B5201 276-284 HTTPILCGA 134 B5801 278-286 TPILCGAQY 135B3501, B4403, Cw0401, Cw0702 279-287 PILCGAQYR 136 A3101 280-288ILCGAQYRI 137 A0201, A0205, A3, B62, B5101

TABLE 28 amino acid sequence HLA position sequence No. subtype 285-293QYRIHTHGV 138 A24, Cw0401 286-294 YRIHTHGVF 139 B14, B2702,B2705, B5201, Cw0301 287-295 RIHTHGVFR 140 A3, A1101, A3101, A3302289-297 HTHGVFRGI 141 B5801

TABLE 29 amino acid sequence HLA position sequence No. subtype 292-300GVFRGIQDV 142 A0201, A0205, A3, A68.1, A1101, B3901, B5102, B5201,Cw0602 293-301 VFRGIQDVR 143 A3101 294-302 FRGIQDVRR 144 B2705 295-303RGIQDVRRV 145 B61, B5101, B5102, B5201, Cw0602 298-306 QDVRRVPGV 146B61, B3701 299-307 DVRRVPGVA 147 A68.1, A3302, B7, B8

TABLE 30 amino acid sequence HLA position sequence No. subtype 301-309RRVPGVAPT 148 B14, B2702, B2705, Cw0301 302-310 RVPGVAPTL   7A0205, A24, B7, B3701, B3801, B3901, B3902 303-311 VPGVAPTLV 149B7, B3501, B5102, B5201, Cw0401 306-314 VAPTLVRSA 150 B4403 309-317TLVRSASET 151 A0201, A0205

TABLE 31 amino acid sequence HLA position sequence No. subtype 312-320RSASETSEK 152 A68.1, A1101 313-321 SASETSEKR 153 A3101, A3302 315-323SETSEKRPF 154 B40, B3701, B4403 316-324 ETSEKRPFM 155 B8, B3501 317-325TSEKRPFMC 156 A1, B5801 318-326 SEKRPFMCA 157 B40, B60, B61, B4403319-327 EKRPFMCAY 158 Cw0602, Cw0702

TABLE 32 amino acid sequence HLA position sequence No. subtype 324-332MCAYPGCNK 159 A68.1, A1101 325-333 CAYPGCNKR 160 A1, A68.1,A1101, A3101, A3302 326-334 AYPGCNKRY 161 A24, Cw0401, Cw0702 327-335YPGCNKRYF 162 B3501, B3801, B5201, Cw0401, Cw0702 329-337 GCNKRYFKL 163A24, B14, B60, B7, B8, B3902, Cw0301

TABLE 33 amino acid sequence HLA position sequence No. subtype 332-340KRYFKLSHL 164 B14, B8, B2702, B2705, B3901, B3902, Cw0301, Cw0602334-342 YFKLSHLQM 165 Cw0401 337-345 LSHLQMHSR 166 A68.1, A3302 340-348LQMHSRKHT 167 A0201 , A0205

TABLE 34 amino acid sequence HLA position sequence No. subtype 343-351HSRKHTGEK 168 A68.1 345-353 RKHTGEKPY 169 Cw0702 347-355 HTGEKPYQC 170B8, B5801 349-357 GEKPYQCDF 171 B40, B3701, B4403

TABLE 35 amino acid sequence HLA position sequence No. subtype 351-359KPYQCDFKD 172 B5102 354-362 QCDFKDCER 173 A1, A68.1, A3101, A3302356-364 DFKDCERRF 174 A24, Cw0401 358-366 KDCERRFSR 175 A3101

TABLE 36 amino acid sequence HLA position sequence No. subtype 362-370RRFSRSDQL 176 B2702, B2705, B3901, Cw0301, Cw0602 363-371 RFSRSDQLK 177A1101 364-372 FSRSDQLKR 178 A68.1, A3302 366-374 RSDQLKRHQ 179 A1368-376 DQLKRHQRR 180 A68.1, A3101, A3302

TABLE 37 amino acid sequence HLA position sequence No. subtype 371-379KRHQRRHTG 181 B14 372-380 RHQRRHTGV 182 B14, B3801, B3901 373-381HQRRHTGVK 183 A1101, A3101, B2705 375-383 RRHTGVKPF 184 B2702, B2705,Cw0702 379-387 GVKPFQCKT 185 A68.1

TABLE 38 amino acid sequence HLA position sequence No. subtype 383-391FQCKTCQRK 186 A3, A1101, A3101, B2705 384-392 QCKTCQRKF 187 B62, B8386-394 KTCQRKFSR 188 A1, A3, A68.1, A1101, A3101 387-395 TCQRKFSRS 189B8 389-397 QRKFSRSDH 190 B2702, B2705 390-398 RKFSRSDHL 191 B14, B3901,B3902, Cw0301

TABLE 39 amino acid sequence HLA position sequence No. subtype 391-399KFSRSDHLK 192 A1101, A3101 394-402 RSDHLKTHT 193 A1, B5801 396-404DHLKTHTRT 194 B3801, B3901

TABLE 40 amino acid sequence HLA position sequence No. subtype 401-409HTRTHTGKT 195 B8 406-414 TGKTSEKPF 196 B5201 408-416 KTSEKPFSC 197A0201, B5801 409-417 TSEKPFSCR 198 A1, A68.1, A3302 410-418 SEKPFSCRW199 B40, B4403

TABLE 41 amino acid sequence HLA position sequence No. subtype 412-420KPFSCRWPS 200 B3501, B5102 415-423 SCRWPSCQK 201 A1101 416-424 CRWPSCQKK202 B2702, B2705 417-425 RWPSCQKKF 203 A24, B3801, Cw0401 418-426WPSCQKKFA 204 B5102 419-427 PSCQKKFAR 205 A3302 420-428 SCQKKFARS 206 B8

TABLE 42 amino acid sequence HLA position sequence No. subtype 423-431KKFARSDEL 207 B14, B3901, B3902, Cw0301, Cw0602 424-432 KFARSDELV 208Cw0401 425-433 FARSDELVR 209 A68.1, A1101, A3101, A3302 426-434ARSDELVRH 210 B2702, B2705 427-435 RSDELVRHH 211 A1 428-436 SDELVRHHN212 B3701 429-437 DELVRHHNM 213 B14, B40, B60, B61, B3701, B4403, Cw0301

TABLE 43 amino acid sequence HLA position sequence No. subtype 432-440VRHHNMHQR 214 B2705 433-441 RHHNMHQRN 215 B3801 434-442 HHNMHQRNM 216B3901 436-444 NMHQRNMTK 217 A3, A1101, A3101 437-445 MHQRNMTKL 218B14, B3701, B3901, B3902, Cw0301 439-447 QRNMTKLQL 220 B14, B2702,B2705, B3901, Cw0602 440-448 RNMTKLQLA 221 B61

TABLE 44 amino acid sequence HLA position sequence No. subtype 441-449NMTKLQLAL 222 A0201, A0205, A24, A3, B7, B3902, Cw0602

Preferable examples of the “MHC class I-restricted WT1 peptide” includea peptide comprising any amino acid sequence selected from the followingamino acid sequences:

RMFPNAPYL, (SEQ ID NO: 2) CMTWNQMNL, (SEQ ID NO: 3) ALLPAVPSL,(SEQ ID NO: 5) SLGEQQYSV (SEQ ID NO: 6) and RVPGVAPTL, (SEQ ID NO: 7) anda peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 2, 3, 5, 6 and 7 but containingalteration of amino acid residue(s), and having a CTL inductionactivity, more preferably a peptide of any of the amino acid sequencesselected from SEQ ID NOs: 2, 3, 5, 6 and 7.

The “peptide comprising an amino acid sequence” in the present inventionmeans, as usual, a peptide wherein a further amino acid is added to theN-terminal amino acid and/or C-terminal amino acid of the amino acidsequence. When the “MHC class I-restricted WT1 peptide” in the “cancerantigen peptide A” and “cancer antigen peptide B” is added, a peptidewith addition to the C-terminal side is preferable. When the “MHC classI-restricted WT1 epitope” is added, addition to the C-terminal side ispreferable.

The “peptide comprising an altered amino acid sequence that containsalteration of amino acid residue(s) in the amino acid sequence, andhaving a CTL induction activity” in the present invention is also calledan “altered killer peptide”. The altered killer peptide means a peptidethat consists of an amino acid sequence wherein 1 to 3 amino acids aredeleted, substituted and/or added and binds to MHC class I to induceCTL. The substitution position of the substituted amino acid includesthe 1st-position (N-terminal), the 2nd-position, the 3rd-position or the9th-position for a peptide consisting of 9 amino acid residues. Thenumber of the amino acids to be added (also including insertion) ispreferably 1 or 2, more preferably 1. A preferable addition position isthe C-terminal. The number of the amino acids to be deleted ispreferably 1. In the alteration, the amino acid to be added or aminoacid to be substituted may be a non-natural amino acid other than the 20kinds of amino acids encoded by the gene.

It is known that the amino acid sequence of a peptide bindable to HLAantigen has regularity (binding motif) for each polymorphism of HLAsubtype. For example, as a binding motif of HLA-A24, a peptideconsisting of 8-11 amino acid residues, wherein the 2nd-position aminoacid is Tyr, Phe, Met or Trp, and the C-terminal amino acid is Phe, Leu,Ile, Trp or Met, is known (J. Immunol., 152, p 3913, 1994, J. Immunol.,155, p 4307, 1994, Immunogenetics, 41, p 178, 1995). Therefore, forexample, in the case of a peptide consisting of 9 amino acid residues,the 2nd-position can be substituted by Tyr, Phe, Met or Trp and/or the9th-position can be substituted by Phe, Leu, Ile, Trp or Met, and apeptide having such substitutions is preferable as an altered killerpeptide. Similarly, a peptide consisting of 8-11 amino acid residues,wherein the 2nd-position amino acid is Leu or Met and the C-terminalamino acid is Val or Leu, is known as a binding motif of HLA-A0201.Therefore, for example, in the case of a peptide consisting of 9 aminoacid residues, the 2nd-position can be substituted by Leu or Met and/orthe 9th-position can be substituted by Val or Leu, and a peptide havingsuch substitutions is preferable as an altered killer peptide.

Examples of the altered killer peptide include the following peptides.

RYFPNAPYL (SEQ ID NO: 223) (see WO03/106682), FMFPNAPYL,(SEQ ID NO: 224) RLFPNAPYL, (SEQ ID NO: 225) RMMPNAPYL, (SEQ ID NO: 226)RMFPNAPYV (SEQ ID NO: 227) and YMFPNAPYL (SEQ ID NO: 228)(see WO2009/072610),which are altered killer peptides of RMFPNAPYL (SEQ ID NO: 2);CYTWNQMNL (SEQ ID NO: 4) (see WO02/79253);Xaa-Met-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 229) (wherein Xaa is Seror Ala) and Xaa-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu (SEQ ID NO: 230)(wherein Xaa is Ser, Ala, Abu, Arg, Lys, Orn, Cit, Leu, Phe or Asn) (seeWO2004/026897), which are altered killer peptides of CMTWNQMNL (SEQ IDNO: 3);AYLPAVPSL (SEQ ID NO: 231) (see WO2003/106682), which is an alteredkiller peptide of ALLPAVPSL (SEQ ID NO: 5);FLGEQQYSV (SEQ ID NO: 232),SMGEQQYSV (SEQ ID NO: 233) andSLMEQQYSV (SEQ ID NO: 234) (WO2009/072610), which are altered killerpeptides of SLGEQQYSV (SEQ ID NO: 6); andRYPGVAPTL (SEQ ID NO: 235) (WO2003/106682), which is an altered killerpeptide of RVPGVAPTL (SEQ ID NO: 7).

“R¹” in the present invention is a hydrogen atom, a group represented bythe aforementioned formula (2) or cancer antigen peptide C, preferably agroup represented by the aforementioned formula (2) or cancer antigenpeptide C.

When R¹ is a hydrogen atom, the compound of the formula (1) is acompound represented by the formula (1-1):

wherein X^(a), Y^(a) and cancer antigen peptide A are as defined in theabove for the formula (1), and Cys is a cysteine residue, namely, apeptide.

The compound of the formula (1), wherein R¹ is a hydrogen atom, namely,a peptide represented by the formula (1-1), has a sequence differentfrom a partial sequence of WT1 protein. The requirement of the formula(1), “has a sequence different from a partial sequence of WT1 protein”means that a peptide represented by the formula (1-1) is not a partialpeptide consisting of continuous 8-35 amino acid residues in the aminoacid sequence of human WT1 described in SEQ ID NO: 1.

That is, the compound of the formula (1), wherein R¹ is a hydrogen atom,is not a partial peptide consisting of continuous 8-35 amino acidresidues in the amino acid sequence of human WT1 described in SEQ IDNO: 1. A specific explanation is given by taking a case when the cancerantigen peptide A is a WT1₁₃₈₋₁₄₆ peptide as an example. WT1₁₃₈₋₁₄₆peptide is a partial peptide consisting of continuous 9 amino acidresidues at the 138th-position-146th-position of the amino acid sequenceof human WT1 described in SEQ ID NO: 1, and has an amino acid sequenceof LESQPAIRN (SEQ ID NO: 78). In SEQ ID NO: 1, the 137th-positioncontinuing from the N-terminal side of WT1₁₃₈₋₁₄₆ peptide is C.Therefore, WT1₁₃₇₋₁₄₆ peptide (CLESQPAIRN) (SEQ ID NO: 236) correspondsto a partial peptide consisting of continuous 10 amino acid residues ofthe amino acid sequence of human WT1 described in SEQ ID NO: 1. On theother hand, based on the requirement of the present invention, “thecompound of the formula (1), wherein R¹ is a hydrogen atom, is not apartial peptide consisting of continuous 8-35 amino acid residues in theamino acid sequence of human WT1 described in SEQ ID NO: 1”, in thecompound of the formula (1) wherein R¹ is a hydrogen atom, when thecancer antigen peptide A is WT1₁₃₈₋₁₄₆ peptide (LESQPAIRN) (SEQ ID NO:78), WT1₁₃₇₋₁₄₆ peptide (CLESQPAIRN) (SEQ ID NO: 236) is excluded fromthe compound of the present invention, and therefore, X^(a) and Y^(a)are not simultaneously a single bond.

As a compound of the formula (1) wherein R¹ is a hydrogen atom, apeptide consisting of any amino acid sequence selected from thefollowing amino acid sequences:

CRMFPNAPYL, (SEQ ID NO: 13) CCMTWNQMNL, (SEQ ID NO: 14) CCYTWNQMNL,(SEQ ID NO: 15) CALLPAVPSL, (SEQ ID NO: 16) CSLGEQQYSV (SEQ ID NO: 17)and CRVPGVAPTL (SEQ ID NO: 18)is preferable.

When “R¹” is a group represented by the aforementioned formula (2), acompound of the formula (1) is a compound represented by the formula(1-2):

wherein X^(a), Y^(a) and cancer antigen peptide A are as defined in theabove for the formula (1), and X^(b), Y^(b) and cancer antigen peptide Bare as defined in the above for the formula (2).

“X^(b)” and “Y^(b)” in the present invention mean, independently, asingle bond or a divalent group of peptides consisting of 1-4 amino acidresidues. The sum of the amino acid residue number of X^(b) and that ofY^(b) is an integer of 0-4. For example, an integer of said sum being 0means that X^(b) and Y^(b) are each a single bond. When the sum is aninteger of 4, examples thereof include X^(b) and Y^(b) independentlybeing divalent groups of peptide consisting of 2 amino acid residues,X^(b) being a divalent group of peptide consisting of 3 amino acidresidues and Y^(b) being a divalent group of peptide consisting of 1amino acid residue, X^(b) being a divalent group of peptide consistingof 4 amino acid residues and Y^(b) being a single bond and the like.

The integer of said sum is preferably 0-2, more preferably 0-1, mostpreferably 0. That is, X^(b) and Y^(b) are most preferably single bonds.

When the sum is an integer of 2, examples thereof include X^(b) being adivalent group of peptide consisting of 2 amino acid residues and Y^(b)being a single bond, X^(b) and Y^(b) independently being divalent groupsof peptide consisting of 1 amino acid residue, and X^(b) being a singlebond and Y^(b) being a divalent group of peptide consisting of 2 aminoacid residues.

When the sum is an integer of 1, examples thereof include X^(b) being adivalent group of peptide consisting of 1 amino acid residue and Y^(b)being a single bond, and X^(b) being a single bond and Y^(b) being adivalent group of peptide consisting of 1 amino acid residue. Of these,preferred is X^(b) being a single bond and Y^(b) being an alanineresidue, leucine residues or methionine residue.

The “cancer antigen peptide B” in the present invention is an MHC classI-restricted WT1 peptide consisting of 7-30 amino acid residues. The“MHC class I-restricted WT1 peptide” is as defined above. However, inthe compound represented by the formula (1), cancer antigen peptide Aand cancer antigen peptide B are not simultaneously the same peptide.That is, cancer antigen peptide B is limited by the requirement,“different from cancer antigen peptide A”.

Since cancer antigen peptide A and cancer antigen peptide B are notsimultaneously the same peptide, the compound of the formula (1),wherein R¹ is a group represented by the aforementioned formula (2), isnot a homodimer but a heterodimer, even when X^(a) and X^(b) are thesame and Y^(a) and Y^(b) are the same. Homodimer means a dimer whereinthe same peptide monomers are dimerized, and heterodimer means a dimerwherein different peptide monomers are dimerized.

In cancer antigen peptide B, the amino group of the N-terminal aminoacid is bonded to Y^(b) in the formula (2) (i.e., also bonded to Y^(b)in the formula (1-2)), and the carbonyl group of the C-terminal aminoacid is bonded to the hydroxyl group in the formula (2).

As a compound of the formula (1) wherein R¹ is a group represented bythe formula (2), i.e., a compound of the formula (1-2), a compoundrepresented by the formula (3):

wherein the bond between C and C is a disulfide bond, is preferable.

In the present invention, moreover, when the “cancer antigen peptide B”is an MHC class I-restricted WT1 peptide containing one cysteineresidue, the compound of the formula (1) may be a compound wherein thethioether group in the cancer antigen peptide B is bonded to a thioethergroup in the formula (16):

or to a thioether group of the cysteine residue of the cancer antigenpeptide E.

“X^(d)” and “Y^(d)” in the present invention mean, independently, asingle bond or a divalent group of peptides consisting of 1-4 amino acidresidues. The sum of the amino acid residue number of X^(d) and that ofY^(d) is an integer of 0-4. For example, an integer of said sum being 0means that X^(d) and Y^(d) are each a single bond. When the sum is aninteger of 4, examples thereof include X^(d) and Y^(d) independentlybeing divalent groups of peptide consisting of 2 amino acid residues,X^(d) being a divalent group of peptide consisting of 3 amino acidresidues and Y^(d) being a divalent group of peptide consisting of 1amino acid residue, X^(d) being a divalent group of peptide consistingof 4 amino acid residues and Y^(d) being a single bond and the like.

The integer of said sum is preferably 0-2, more preferably 0-1, mostpreferably 0. That is, X^(d) and Y^(d) are most preferably single bonds.

When the sum is an integer of 2, examples thereof include X^(d) being adivalent group of peptide consisting of 2 amino acid residues and Y^(b)being a single bond, X^(d) and Y^(d) independently being divalent groupsof peptide consisting of 1 amino acid residue, or X^(d) being a singlebond and Y^(d) being a divalent group of peptide consisting of 2 aminoacid residues.

When the sum is an integer of 1, examples thereof include X^(d) being adivalent group of peptide consisting of 1 amino acid residue and Y^(d)being a single bond, and X^(d) being a single bond and Y^(d) being adivalent group of peptide consisting of 1 amino acid residue. Of these,preferred is X^(d) being a single bond and Y^(d) being an alanineresidue, leucine residues or methionine residue.

The “cancer antigen peptide D” in the present invention is an MHC classII-restricted WT1 peptide consisting of 7-30 amino acid residues. In theformula (16), an amino group of the N-terminal amino acid of the cancerantigen peptide D binds to Y^(d) in the formula (16), and a carbonylgroup of the C-terminal amino acid binds to a hydroxyl group in theformula (16).

In the present invention, “MHC class II-restricted” means the propertyto induce helper T cell by binding to an MHC class II molecule, and isas defined for the below-mentioned “cancer antigen peptide C”.

HLA corresponding to the MHC class II-molecule is classified intosubtypes of HLA-DR, DQ and DP and the like. Preferable examples of the“MHC class II-restricted” include HLA-DR-restricted, HLA-DQ-restrictedand HLA-DP-restricted.

Therefore, the “MHC class II-restricted WT1 peptide” in the presentinvention is a peptide that binds to an MHC class II antigen in vitroand/or in vivo and induces helper T cells. The number of the amino acidresidues of the “MHC class II-restricted WT1 peptide” is 7-30,preferably 14-30.

As the “cancer antigen peptide D”, like the amino acid sequence recitedin the below-mentioned “cancer antigen peptide C”, a peptide consistingof any amino acid sequence selected from the following amino acidsequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQAYMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH,(SEQ ID NO: 23) CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24) andWAPVLDFAPPGASAYGSL (SEQ ID NO: 244)can be mentioned.

In the compound of the formula (1), moreover, when the “cancer antigenpeptide B” is an MHC class I-restricted WT1 peptide containing onecysteine residue, as a compound wherein the thioether group in thecancer antigen peptide B is bonded to a thioether group in the formula(16), preferably, a compound represented by the formula (15):

wherein the bond between C and C is a disulfide bond, can be mentioned.

In the present invention, the “cancer antigen peptide E” is an MHC classII-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue, and is as defined for the “MHC classII-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue” in the below-mentioned “cancer antigenpeptide C”.

As the “cancer antigen peptide E”, like the amino acid sequence recitedin the below-mentioned “cancer antigen peptide C”, a peptide consistingof any amino acid sequence selected from the following amino acidsequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH(SEQ ID NO: 23) and CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24)can be mentioned.

When R¹ is cancer antigen peptide C, the thioether group of the cysteineresidue of cancer antigen peptide C is bonded to the thioether group inthe formula (1).

The cancer antigen peptide C is an MHC class I-restricted WT1 peptideconsisting of 7-30 amino acid residues containing one cysteine residueor an MHC class II-restricted WT1 peptide consisting of 7-30 amino acidresidues containing one cysteine residue.

In the “MHC class I-restricted WT1 peptide consisting of 7-30 amino acidresidues containing one cysteine residue” in the present invention, theamino acid sequence of the peptide only needs to contain at least onecysteine residue. The number of the cysteine residues to be contained ispreferably 1-3, more preferably 1-2, most preferably 1. The “MHC classI-restricted WT1 peptide” is as defined above. Also, the compound of theformula (1) wherein R¹ is “an MHC class I-restricted WT1 peptideconsisting of 7-30 amino acid residues containing one cysteine residue”is not a homodimer but a heterodimer.

Preferable examples of the “MHC class I-restricted WT1 peptideconsisting of 7-30 amino acid residues containing one cysteine residue”include peptides described in Tables 45-52. In each Table, the“position” means the position in the amino acid sequence of human WT1described in SEQ ID NO: 1.

TABLE 45 amino acid sequence HLA position sequence No. subtype 17-25SLGGGGGCA 31 B62 18-26 LGGGGGCAL 32 B60, B7, B3801, B5101, B5102 20-28GGGGCALPV 33 B61, B5101, B5102, B5201 23-31 GCALPVSGA 34 B40, B61 24-32CALPVSGAA 35 B40, B5102, Cw0301

TABLE 46 amino acid sequence HLA position sequence No. subtype  80-88GAEPHEEQC 51 A1  81-89 AEPHEEQCL 52 A0205, B40, B60, B61, B3701, B4403 82-90 EPHEEQCLS 53 B3501, B5101  83-91 PHEEQCLSA 54 B3801  84-92HEEQCLSAF 55 B40, B3701, B4403, Cw0702  85-93 EEQCLSAFT 56B40, B60, B61, B3701, B4403  86-94 EQCLSAFTV 57 A0201, B62, B5201  88-96CLSAFTVHF 58 A3, B62  99-107 QFTGTAGAC 63 Cw0401 100-108 FTGTAGACR 64A68.1, A1101, A3101, A3302

TABLE 47 amino acid sequence HLA position sequence No. subtype 101-109TGTAGACRY 65 B62, B4403, Cw0702 104-112 AGACRYGPF 66 B4403, B5201107-115 CRYGPFGPP 67 B2702 130-138 NAPYLPSCL 75 A24, B60, B7, B8, B3902,B5101, B5102, Cw0301, Cw0602, Cw0702 136-144 SCLESQPAI 76 B8, B3901,B5102, Cw0301 137-145 CLESQPAIR 77 A1, A3, A68.1, A1101, A3101, A3302194-202 SVPPPVYGC 97 A0205, A3

TABLE 48 amino acid sequence HLA position sequence No. subtype 202-210CHTPTDSCT  98 B3801 204-212 TPTDSCTGS  99 B5101 206-214 TDSCTGSQA 100B40, B61, B3701 207-215 DSCTGSQAL 101 A24, A3302, B60, B7, B8,B3501, B3901, B3902, Cw0602 208-216 SCTGSQALL 102 B60, B7, B8,B3701, B3801, B3901, B3902 209-217 CTGSQALLL 103 B60, B7, B3701, B3902227-235 YQMTSQLEC 114 A0201, A0205, B62 228-236 QMTSQLECM 115 A0201

TABLE 49 amino acid sequence HLA position sequence No. subtype 230-238TSQLECMTW 116 B5801 232-240 QLECMTWNQ 117 A1 233-241 LECMTWNQM 118B40, B60, B61, B3701, B4403 235-243 CMTWNQMNL   3 A0201, A0205,A24, A3, B7 276-284 HTTPILCGA 134 B5801 278-286 TPILCGAQY 135B3501, B4403, Cw0401, Cw0702 279-287 PILCGAQYR 136 A3101 280-288ILCGAQYRI 137 A0201, A0205, A3, B62, B5101

TABLE 50 amino acid sequence HLA position sequence No. subtype 317-325TSEKRPFMC 156 A1, B5801 318-326 SEKRPFMCA 157 B40, B60, B61, B4403319-327 EKRPFMCAY 158 Cw0602, Cw0702 324-332 MCAYPGCNK 159 A68.1, A1101325-333 CAYPGCNKR 160 A1, A68.1, A1101, A3101, A3302 326-334 AYPGCNKRY161 A24, Cw0401, Cw0702 327-335 YPGCNKRYF 162 B3501, B3801,B5201, Cw0401, Cw0702 329-337 GCNKRYFKL 163 A24, B14, B60,B7, B8, B3902, Cw0301 347-355 HTGEKPYQC 170 B8, B5801 349-357 GEKPYQCDF171 B40, B3701, B4403

TABLE 51 amino acid sequence HLA position sequence No. subtype 351-359KPYQCDFKD 172 B5102 354-362 QCDFKDCER 173 A1, A68.1, A3101, A3302356-364 DFKDCERRF 174 A24, Cw0401 358-366 KDCERRFSR 175 A3101 379-387GVKPFQCKT 185 A68.1 383-391 FQCKTCQRK 186 A3, A1101, A3101, B2705384-392 QCKTCQRKF 187 B62, B8 386-394 KTCQRKFSR 188 A1, A3, A68.1,A1101, A3101 387-395 TCQRKFSRS 189 B8

TABLE 52 amino acid sequence HLA position sequence No. subtype 408-416KTSEKPFSC 197 A0201, B5801 409-417 TSEKPFSCR 198 A1, A68.1, A3302410-418 SEKPFSCRW 199 B40, B4403 412-420 KPFSCRWPS 200 B3501, B5102415-423 SCRWPSCQK 201 A1101 416-424 CRWPSCQKK 202 B2702, B2705 417-425RWPSCQKKF 203 A24, B3801, Cw0401 418-426 WPSCQKKFA 204 B5102 419-427PSCQKKFAR 205 A3302 420-428 SCQKKFARS 206 B8

More preferable examples of the “MHC class I-restricted WT1 peptideconsisting of 7-30 amino acid residues containing one cysteine residue”include a peptide comprising the following amino acid sequence:

CMTWNQMNL (SEQ ID NO: 3)

and a peptide comprising an altered amino acid sequence, which is theamino acid sequence of SEQ ID NO: 3 but containing alteration of aminoacid residue(s), and having a CTL induction activity. Said “containingthe amino acid sequence” and “peptide comprising an altered amino acidsequence containing alteration of amino acid residue(s) in an amino acidsequence, and having a CTL induction activity” are as defined above.Most preferably, a peptide consisting of any amino acid sequenceselected from the following amino acid sequences:

CMTWNQMNL (SEQ ID NO: 3) and CYTWNQMNL, (SEQ ID NO: 4)can be mentioned.

As the compound of the formula (1) wherein R′ is “an MHC classI-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue”, a compound represented by the formula(4):

wherein the bond between C and C is a disulfide bond, or a compoundrepresented by the formula (5):

wherein the bond between C and C is a disulfide bond, is preferable. Ofthese, a compound represented by the formula (5) is more preferable.

In the present invention, moreover, when a peptide consisting of 1-4amino acid residues containing one cysteine residue is further bonded tothe N-terminal of the “cancer antigen peptide C” which is an MHC classI-restricted WT1 peptide, the compound of the formula (1) may be acompound wherein the thioether group of the cysteine residue of thepeptide bonded to the N-terminal of the cancer antigen peptide C isbonded to a thioether group in the formula (16):

or to a thioether group of the cysteine residue of the cancer antigenpeptide E.

The “X^(d)”, “Y^(d)”, “cancer antigen peptide D” and “cancer antigenpeptide E” in the present invention are as defined for theaforementioned “X^(d)”, “Y^(d)”, “cancer antigen peptide D” and “cancerantigen peptide E”.

In the present invention, the peptide consisting of 1-4 amino acidresidues containing one cysteine residue, which is bonded to theN-terminal of the “cancer antigen peptide C” which is an MHC classI-restricted WT1 peptide, is preferably a dipeptide consisting of CA.

In the compound of the formula (1), when a peptide consisting of 1-4amino acid residues containing one cysteine residue is further bonded tothe N-terminal of the “cancer antigen peptide C” which is an MHC classI-restricted WT1 peptide, the compound wherein the thioether group ofthe cysteine residue of the peptide bonded to the N-terminal of thecancer antigen peptide C is bonded to a thioether group in the formula(16) is preferably a compound represented by the formula (14):

wherein the bond between C and C is a disulfide bond.

In addition, in the compound of the formula (1), when a peptideconsisting of 1-4 amino acid residues containing one cysteine residue isfurther bonded to the N-terminal of the “cancer antigen peptide C” whichis an MHC class I-restricted WT1 peptide, the compound wherein thethioether group of the cysteine residue of the peptide bonded to theN-terminal of the cancer antigen peptide C is bonded to a thioethergroup in the “cancer antigen peptide E” is preferably a compoundrepresented by the formula (12):

wherein the bond between C and C is a disulfide bond.

In the “MHC class II-restricted WT1 peptide consisting of 7-30 aminoacid residues containing one cysteine residue” in the present invention,the amino acid sequence of the peptide only needs to contain at leastone cysteine residue. The number of the cysteine residues to becontained is preferably 1-3, more preferably 1-2, most preferably 1.

In the present invention, “MHC class II-restricted” means the propertyto induce helper T cell by binding to an MHC class II molecule.

HLA corresponding to the MHC class II-molecule is classified intosubtypes of HLA-DR, DQ and DP and the like. Preferable examples of the“MHC class II-restricted” include HLA-DR-restricted, HLA-DQ-restrictedand HLA-DP-restricted.

Therefore, the “MHC class II-restricted WT1 peptide” in the presentinvention is a peptide that binds to an MHC class II antigen in vitroand/or in vivo and induces helper T cells. The number of the amino acidresidues of the “MHC class II-restricted WT1 peptide” is 7-30,preferably 14-30.

Examples of the “MHC class II-restricted WT1 peptide consisting of 7-30amino acid residues containing one cysteine residue” include thepeptides described in Table 53. In each Table, the “position” means aposition in the amino acid sequence of human WT1 described in SEQ ID NO:1.

TABLE 53 sequence position amino acid sequence No. 117-139PSQASSGQARMFPNAPYLPSCLE 237 122-140 SGQARMFPNAPYLPSCLES  11 202-233CHTPTDSCTGSQALLLRTPYSSDNLYQMTSQL   9 328-349 PGCNKRYFKLSHLQMHSRKHTG  10421-441 CQKKFARSDELVRHHNMHQRN 219

As the “MHC class II-restricted WT1 peptide consisting of 7-30 aminoacid residues containing one cysteine residue”, a peptide comprising anyamino acid sequence selected from the following amino acid sequences:

SGQARMFPNAPYLPSCLES (SEQ ID NO: 11) and PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10)ora peptide comprising an altered amino acid sequence, which is any aminoacid sequence selected from SEQ ID NOs: 10-11 but containing alterationof amino acid residue(s), and having a helper T cell induction activityis preferable.

The “peptide comprising an amino acid sequence” means, as mentionedabove, a peptide wherein a further amino acid is added to the N-terminalamino acid and/or C-terminal amino acid of the amino acid sequence. Whenadded to the “MHC class II-restricted WT1 peptide containing onecysteine residue”, the addition may be made to the N-terminal sideand/or C-terminal side.

The “peptide comprising an altered amino acid sequence containingalteration of amino acid residue(s) in the amino acid sequence, andhaving a helper T cell induction activity” in the present invention isalso called an “altered helper peptide”. The altered helper peptidemeans a peptide that consists of an amino acid sequence wherein 1 to 3amino acids are deleted, substituted and/or added and binds to MHC classII to induce helper T cell. The number of the amino acids to be added(also including insertion) is preferably 1-3. The number of the aminoacids to be deleted is preferably 1-5. In the alteration, the amino acidto be added or amino acid to be substituted may be a non-natural aminoacid other than the kinds of amino acids encoded by the gene.

As the altered helper peptide, for example, the following peptides canbe mentioned:

SGQAYMFPNAPYLPSCLES (SEQ ID NO: 12) (see patent document 6),

SGQAYMFPNAPYLPSC (SEQ ID NO: 19) and

SGQAYMFPNAPYLPSC (SEQ ID NO: 25), which are altered helper peptide ofSGQARMFPNAPYLPSCLES (SEQ ID NO: 11); and

GCNKRYFKLSHLQMHSRK (SEQ ID NO: 20),

PGCNKRYFKLSHLQMHSRKH (SEQ ID NO: 21),

CNKRYFKLSHLQMHSRK (SEQ ID NO: 22),

CNKRYFKLSHLQMHSRKH (SEQ ID NO: 23) and

CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24), which are altered helper peptideof PGCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 10).

As the “MHC class II-restricted WT1 peptide consisting of 7-30 aminoacid residues containing one cysteine residue”, a peptide consisting ofany amino acid sequence selected from the following amino acidsequences:

SGQARMFPNAPYLPSC, (SEQ ID NO: 19) SGQAYMFPNAPYLPSC, (SEQ ID NO: 25)SGQARMFPNAPYLPSCLES, (SEQ ID NO: 11) SGQAYMFPNAPYLPSCLES,(SEQ ID NO: 12) PGCNKRYFKLSHLQMHSRK, (SEQ ID NO: 20)PGCNKRYFKLSHLQMHSRKH, (SEQ ID NO: 21) PGCNKRYFKLSHLQMHSRKHTG,(SEQ ID NO: 10) CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH(SEQ ID NO: 23) and CNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24)is more preferable.

As the compound of the formula (1) wherein R¹ is “an MHC classII-restricted WT1 peptide consisting of 7-30 amino acid residuescontaining one cysteine residue”, a compound represented by the formula(6):

wherein the bond between C and C is a disulfide bond, a compoundrepresented by the formula (7):

wherein the bond between C and C is a disulfide bond, a compoundrepresented by the formula (8):

wherein the bond between C and C is a disulfide bond, and a compoundrepresented by the formula (9):

wherein the bond between C and C is a disulfide bond, are preferable.

The present invention also provides a composition comprising thecompound of the present invention and one or more MHC classII-restricted WT1 peptides.

Examples of the compound of the present invention to be contained in thecomposition of the present invention include a compound represented bythe formula (3):

wherein the bond between C and C is a disulfide bond, a compoundrepresented by the formula (4):

wherein the bond between C and C is a disulfide bond, and a compoundrepresented by the formula (5):

wherein the bond between C and C is a disulfide bond.

Examples of the MHC class II-restricted WT1 peptide to be contained inthe composition of the present invention include the following aminoacid sequences:

CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH, (SEQ ID NO: 23)CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24) WAPVLDFAPPGASAYGSL,(SEQ ID NO: 244) CWAPVLDFAPPGASAYGSL (SEQ ID NO: 242) andWAPVLDFAPPGASAYGSLC. (SEQ ID NO: 243)

The present invention also provides a synthesis method of a compoundwherein two different MHC class I-restricted WT1 peptide and MHC classII-restricted WT1 peptide, or two different MHC class I-restricted WT1epitope and MHC class II-restricted WT1 epitope are each bonded via adisulfide bond. The method of the present invention includes thefollowing steps (1)-(3).

In step (1) of the present invention, a peptide wherein a carbonyl groupof the C-terminal amino acid of C(Mmt)A and the N-terminal amino groupof the cancer antigen peptide C are bonded is synthesized by usingFmoc-C(Mmt)A-SBn and cancer antigen peptide C.

The “cancer antigen peptide C” is as defined for the aforementioned“cancer antigen peptide C”. “Fmoc” is a 9-fluorenylmethoxycarbonylgroup. “Mmt” is a monomethoxytrityl group. “SBn” is a thiobenzyl group.

In step (2) of the present invention, a peptide wherein a thioethergroup of the cysteine residue of the cancer antigen peptide C in thepeptide obtained in the aforementioned step (1) and a thioether group ofthe cysteine residue bonded to the N-terminal of cancer antigen peptideA are bonded is synthesized by using the peptide obtained in theaforementioned step (1) and cancer antigen peptide A wherein onecysteine residue protected by Npys group is bonded to the N-terminal.

The “cancer antigen peptide A” is as defined for the aforementioned“cancer antigen peptide A”. “Npys” is a 3-nitro-2-pyridylthio group.

In step (3) of the present invention, a peptide wherein a thioethergroup of the cysteine residue bonded to the N-terminal of the cancerantigen peptide A in the peptide obtained in the aforementioned step(2), and a thioether group of the cysteine residue of the cancer antigenpeptide D are bonded, is synthesized by using the peptide obtained inthe aforementioned step (2) and cancer antigen peptide D containing acysteine residue protected by Spy group.

The “cancer antigen peptide D” is as defined for the aforementioned“cancer antigen peptide D”. “SPy” is a 2-pyridylsulfide group.

The compound and peptide of the present invention, and peptides to beintermediates therefor can be produced according to the method describedin the Examples of the present specification or a method to be generallyused for the peptide synthesis. Examples of the production methodinclude the methods described in the documents (Peptide Synthesis,Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc.,New York, 1976; peptide synthesis, Maruzen Co., LTD., 1975; Basics andExperiment of Peptide Synthesis, Maruzen Co., LTD., 1985; Development ofPharmaceutical Product subsequent vol. 14, Peptide Synthesis, HirokawaShoten, 1991) and the like.

Examples thereof include a production method by a solid phasesynthesizer using Fmoc method or Boc method, and a production method bysequential condensation of Boc-amino acid or Z-amino acid by liquidphase synthesis process (Fmoc is a 9-fluorenylmethoxycarbonyl group, Bocis a t-butoxycarbonyl group, and Z is a benzyloxycarbonyl group).

In the intermediate for the production of the compound of the presentinvention, a functional group such as an amino group, a carboxy group, amercapto group and the like can be protected by a suitable protectinggroup or deprotected as necessary using protection and deprotectiontechniques. As preferable protecting groups, protection method anddeprotection method are described in detail in “Protective Groups inOrganic Synthesis 2nd Edition (John Wiley & Sons, Inc.; 1990)” and thelike. Examples of the mercapto-protecting group include anacetamidomethyl group, a trityl group and the like.

When the compound of the present invention has a disulfide bond, thedisulfide bond can be formed between two different peptides containing acysteine residue or between peptide containing a cysteine residue andcysteine according to a method generally used for peptide chemistry.Examples of the formation method of the disulfide bond include themethods described in the documents (Peptide Synthesis, Interscience, NewYork, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976;peptide synthesis, Maruzen Co., LTD., 1975; Basics and Experiment ofpeptide synthesis, Maruzen Co., LTD., 1985; Development ofPharmaceutical Product sequential vol. 14, Peptide Synthesis, HirokawaShoten, 1991) and the like.

Specifically, when a peptide contains one cysteine residue, a compoundhaving a disulfide bond (disulfide compound) can be produced by removingall protecting groups including the mercapto-protecting group on thecysteine side chain and oxidizing in an inert solvent. In addition, itcan be produced by mixing two intermediates having a mercapto group in asuitable solvent to allow oxidation. As a method for the oxidation, aknown method for forming a disulfide bond in general peptide synthesiscan be selected as appropriate. For example, iodine oxidation, a methodincluding air oxidation reaction under alkali conditions, a method forforming a disulfide bond by adding an oxidant under alkaline or acidicconditions and the like can be mentioned. Here, as the oxidant, iodine,dimethyl sulfoxide (DMSO), potassium ferricyanide and the like can bementioned. As the solvent, water, acetic acid, methanol, chloroform,DMF, DMSO and the like, or a mixture thereof can be used. An oxidationreaction often affords a mixture of symmetric, asymmetric disulfidecompounds. The object asymmetric disulfide compound can be obtained bypurifying by various chromatography, recrystallization and the like.Alternatively, an intermediate having an activated mercapto group and anintermediate having a mercapto group are mixed to form a selectivedisulfide bond. As the intermediate having an activated mercapto group,a mercapto group bonded with an Npys group (3-nitro-2-pyridinesulphenylgroup) and the like can be mentioned. Alternatively, one intermediateand, for example, 2,2′-dithiobis(5-nitropyridine) are mixed in advanceto activate the mercapto group, and then the other intermediate isadded, whereby a selective disulfide bond can be formed (TetrahedronLetters. Vol. 37. No. 9, pp. 1347-1350).

Also, when two or more cysteine residues are contained in the peptide, amethod similar to the aforementioned method can be used. In this case,an isomer with a different manner of disulfide bond is obtained. A dimerwherein a disulfide bond is formed between the object cysteine residuescan be obtained by using a particular combination of the cysteine sidechain-protecting groups. As the aforementioned combination of protectinggroups, MeBzl (methylbenzyl) group and Acm (acetamidomethyl) group, Trt(trityl) group and Acm group, Npys (3-nitro-2-pyridylthio) group and Acmgroup, S-Bu-t (S-tert-butyl) group and Acm group and the like can bementioned. For example, in the case of a combination of MeBzl group andAcm group, a method of forming a disulfide bond between cysteineresidues protected Acm group, which includes removing protecting groupother than MeBzl group and cysteine side chain, subjecting a solutioncontaining a peptide monomer to air oxidation reaction to form adisulfide bond between the deprotected cysteine residues, and thenperforming deprotection with iodine and oxidation and the like can bementioned.

The obtained compound, peptide and intermediate of the present inventioncan be purified according to a method known to those of ordinary skillin the art and a method generally used for peptide chemistry. Forexample, they can be purified by various chromatography (e.g., silicagel column chromatography, ion exchange column chromatography, gelfiltration, reversed-phase chromatography), recrystallization and thelike. For example, as the recrystallization solvent, alcohol solventssuch as methanol, ethanol, 2-propanol and the like, ether solvents suchas diethyl ether and the like, ester solvents such as ethyl acetate andthe like, aromatic hydrocarbon solvents such as benzene, toluene and thelike, ketone solvents such as acetone and the like, hydrocarbon solventssuch as hexane and the like, aprotonic solvents such asdimethylformamide, acetonitrile and the like, water, a mixed solventthereof and the like can be used. As other purified by methods, themethods described in Jikken Kagaku Kouza (The Chemical Society of Japaned., Maruzen) vol. 1 etc., and the like can be used.

The purified by methods of the disulfide compound are described in thedocuments (Peptide Synthesis, Interscience, New York, 1966; TheProteins, Vol. 2, Academic Press Inc., New York, 1976; peptidesynthesis, Maruzen Co., LTD., 1975; Basics and Experiment of PeptideSynthesis, Maruzen Co., LTD., 1985; Development of PharmaceuticalProduct sequential vol. 14 synthesis, Hirokawa Shoten, 1991) and thelike. Among these, HPLC is preferable.

When the compound of the present invention has one or more asymmetricpoints, it can be produced according to a general method and using astarting material having the asymmetric points (amino acid). To increasethe optical purity of the compound of the present invention, moreover,optical resolution and the like may be performed at a suitable stage ofthe production step. As the optical resolution method, for example, adiastereomer method including forming a salt of the compound of thepresent invention or an intermediate thereof with an optically activeacid (e.g., monocarboxylic acids such as mandelic acid,N-benzyloxyalanine, lactic acid and the like, dicarboxylic acids such astartaric acid, o-diisopropylidenetartaric acid, malic acid and the like,or sulfonic acids such as camphorsulfonic acid, bromocamphorsulfonicacid and the like) in an inert solvent (e.g., alcohol solvents such asmethanol, ethanol, 2-propanol and the like, ether solvents such asdiethyl ether and the like, ester solvents such as ethyl acetate and thelike, hydrocarbon solvents such as toluene and the like, aprotonicsolvents such as acetonitrile and the like, and a mixed solvent thereof)can be used. When the compound of the present invention or intermediatehas an acidic functional group such as carboxy group and the like,optical resolution can also be performed by forming a salt with anoptically active amine (e.g., organic amine such as α-phenethylamine,kinin, quinidine, cinchonidine, cinchonine, strychnine and the like).

The temperature for forming a salt is selected from the range of roomtemperature to the boiling point of the solvent. To improve the opticalpurity, it is desirable to once raise the temperature to around theboiling point of the solvent. When the precipitated salt is collected byfiltration, it can be cooled as necessary to increase the yield. Asuitable amount of the optically active acid, or amine to be used iswithin the range of about 0.5-about 2.0 equivalents, preferably about 1equivalent, relative to the substrate. Where necessary, the crystals maybe recrystallized in an inert solvent (e.g., alcohol solvents such asmethanol, ethanol, or 2-propanol and the like, ether solvents such asdiethyl ether and the like, ester solvents such as ethyl acetate and thelike, hydrocarbon solvents such as toluene and the like, aprotonicsolvents such as acetonitrile and the like, and a mixed solvent thereof)to also afford an optically active salt with high purity. Wherenecessary, optically resolved salt may be treated with an acid or baseby a general method to give a free form.

Examples of the “pharmaceutically acceptable salt” in the presentinvention include acid addition salt and base addition salt. Examples ofthe acid addition salt include inorganic acid salts such ashydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphateand the like, and organic acid salts such as citrate, oxalate, acetate,formate, propionate, benzoate, trifluoroacetate, maleate, tartrate,methanesulfonate, benzenesulfonate, p-toluenesulfonate and the like.Examples of the base addition salt include salts with inorganic basesuch as sodium salt, potassium salt, calcium salt, magnesium salt,ammonium salt and the like, salts with organic base such astriethylammonium salt, triethanolammonium salt, pyridinium salt,diisopropylammonium salt etc., and the like, furthermore, amino acidsalts of basic or acidic amino acids such as arginine, aspartic acid,glutamic acid and the like.

The present invention also encompasses hydrates, solvates such asethanol solvate and the like of the compound of the present invention ora pharmaceutically acceptable salt thereof. Furthermore, the presentinvention encompasses any stereoisomers such as any diastereomer,enantiomer and the like and any crystals in any embodiments, of thecompound represented by the formula (1), that can be present.

In general, in the production of peptide, various byproducts such asamino acid-defective peptide, peptide degraded by hydrolysis, oxidationand the like, peptide with racemized amino acid and the like occur in astep of condensing optically active α-amino acid, a step of removingvarious protecting groups, a step of cleaving peptide from a resin andthe like. At a laboratory scale, various chromatographys (e.g., silicagel column chromatography, ion exchange column chromatography, gelfiltration, and reversed-phase chromatography) are combined to removesuch impurities, whereby peptide and a compound with high purity can beobtained. However, it is not easy to obtain peptide and a compound withhigh purity at an industrial scale to provide pharmaceutical products.

The compound of the present invention has physicochemical properties toallow mass production of a drug substance for pharmaceutical products.Specifically, it has high solubility, is superior in the stability in asolution, is hard to become gelled when concentrated and the like, andthe compound can be produced easily as a drug substance with high purityat a large scale by a purified by step using column chromatography suchas reversed-phase HPLC and the like.

The thus-produced compound of the present invention is superior in thestability to oxidant and the like in a solution, since the cysteineresidues form a disulfide bond and the like, and retains given qualityas a drug substance of medicaments and efficient CTL induction activity.

The compound of the present invention is useful as an active ingredientof a CTL induction agent for cancer immunotherapy, an active ingredientof a cancer vaccine, or an active ingredient of a pharmaceuticalcomposition. That is, the compound of the present invention has, asshown in the Examples of the present specification, superiorimmunogenicity and can efficiently show a superior CTL inductionactivity. In addition, CTL induced by the compound of the presentinvention can surprisingly recognize natural type partial peptide of WT1inherently present in cancer cells.

The CTL induction activity can be detected by measuring the number ofCTL by the HLA tetramer method (Int. J. Cancer: 100, 565-570 (2002)) orlimiting dilution method (Nat. Med.: 4, 321-327 (1998)). Alternatively,for example, HLA-A24-restricted CTL induction activity can be examinedby using the HLA-A24 model mouse described in WO 02/47474 and Int. J.Cancer: 100, 565-570 (2002) and the like.

Therefore, the compound of the present invention can be used as atherapeutic drug or prophylactic drug (recurrence preventive drug) forcancer expressing WT1 gene or cancer associated with an increase in theWT1 gene expression level. Examples of the cancer include hematologiccancer such as leukemia, myelodysplastic syndrome, multiple myeloma,malignant lymphoma and the like, and solid tumor such as gastric cancer,colorectal cancer, lung cancer, breast cancer, germ cell cancer, livercancer, skin cancer, urinary bladder cancer, prostate cancer, uterinecancer, cervical cancer, ovarian cancer, brain tumor and the like.

The compound of the present invention or a pharmaceutically acceptablesalt thereof can be an active ingredient of a CTL induction agent forcellular immunotherapy of cancer, an active ingredient of a cancervaccine or/and an active ingredient of a pharmaceutical composition, byformulating each compound or salt in a suitable form.

The compound of the present invention can be administered together witha carrier acceptable as a medicament such as a suitable adjuvant so thatits cellular immunity will be established effectively. As the adjuvant,those described in a document (Clin. Microbiol. Rev., 7: 277-289, 1994)and the like are applicable. Specifically, fungus-derived components,GM-CSF, cytokines such as interleukin-2, interleukin-7, interleukin-12and the like, plant-derived components, marine organism-derivedcomponents, mineral gel such as aluminum hydroxide, lysolecithin,surfactants such as pluronic polyol, polyanion, peptide, oil emulsion(emulsion preparation) and the like can be mentioned. As thefungus-derived components, lipid A, monophosphoryl lipid A, which is aderivative thereof, dead bacteria (Mycobacterium bacteria such as BCGbacteria and the like), bacterium-derived proteins, polynucleotides,Freund's

Incomplete Adjuvant, Freund's Complete Adjuvant, cell wall skeletoncomponents (e.g., BCG-CWS and the like), trehalose dimycolate (TDM) andthe like can be mentioned.

In addition, the compound of the present invention can also beadministered in the form of a liposome preparation, a particulatepreparation including binding to beads with a diameter of several apreparation including binding to a lipid and the like.

Furthermore, the compound of the present invention (conjugate) can beadministered together with an MHC class II-restricted WT1 peptide(namely, helper peptide). As a method for co-administration, a conjugateand a helper peptide may be individually administered. A cocktailpreparation (cocktail agent, cocktail) containing a conjugate and ahelper peptide in a single pharmaceutical composition is morepreferable. The cocktail preparation contains a conjugate capable ofproducing MHC class I-restricted WT1 peptide (i.e., killer peptide) andMHC class II-restricted WT1 peptide (namely, helper peptide). Therefore,by administering the cocktail preparation containing a helper peptide,as a cancer vaccine for cancer immunotherapy, helper T cells importantfor functional promotion of other T cells including CTL can also beactivated, and function and efficacy (cellular immunocompetence and thelike) of the conjugate can be improved.

The MHC class II-restricted WT1 peptide (namely, helper peptide) is asdescribed in the DESCRIPTION. Examples of the helper peptide for thecocktail preparation include the following amino acid sequences:

CNKRYFKLSHLQMHSRK, (SEQ ID NO: 22) CNKRYFKLSHLQMHSRKH, (SEQ ID NO: 23)CNKRYFKLSHLQMHSRKHTG, (SEQ ID NO: 24) WAPVLDFAPPGASAYGSL,(SEQ ID NO: 244) CWAPVLDFAPPGASAYGSL (SEQ ID NO: 242) and

WAPVLDFAPPGASAYGSLC. (SEQ ID NO: 243)Of these, WAPVLDFAPPGASAYGSL (SEQ ID NO: 244) is preferable.

It could be confirmed that the cocktail preparation shows improvedefficacy as a cancer vaccine such as cellular immunocompetence and thelike, as shown in, for example, Examples and Experimental Examples inthe DESCRIPTION.

While the dose of the compound of the present invention in thepreparation can be appropriately controlled according to the treatmentobject disease, age and body weight of the patients and the like, it isgenerally 0.0001 mg-1000 mg, preferably 0.001 mg-1000 mg, morepreferably 0.1 mg-10 mg.

As the administration method, intradermal administration, subcutaneousadministration, intramuscular administration, intravenousadministration, transdermal administration and the like can bementioned. Intradermal administration and subcutaneous administrationare preferable since they efficiently induce CTL. While theadministration frequency and administration intervals can beappropriately controlled according to the prophylaxis or treatment ofobject disease, and individual difference in patients, it is generallymultiple times, and administration once per several days to severalmonths is preferable.

By administering a pharmaceutical composition containing such compoundof the present invention as an active ingredient to WT1 positivepatients, a method for the prophylaxis or treatment of cancer can beprovided.

EXAMPLES

The present invention is specifically explained in the following byreferring to Examples, to which, however, the invention is not limited.

Example 1

Synthesis of the compound represented by the formula (5):

wherein the bond between C and C is a disulfide bond.

Step 1. Synthesis of H-Cys(Npys)-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH

(Synthesis of C(Npys)RMFPNAPYL)

Using Fmoc-Leu-Alko-resin (Alko is p-alkoxybenzylalcohol), 282 mg,(manufactured by Watanabe Chemical; 0.71 mmol/g, 0.2 mmol) as a startingmaterial, the peptide chain was assembled by solid phase synthesisaccording to Fmoc/tBu method. Solid phase synthesis was performed usingCS336X peptide synthesizer manufactured by CS Bio, and deprotection ofFmoc group was performed by treatment with a DMF solution of 20%piperidine for 5 min and for 20 min. Coupling of protected amino acidwas performed by reaction with a DMF solution of 1.05 mmol of protectedamino acid, 1 mmol HBTU and 2 mmol DIPEA for 1 hr. The obtained resinwas washed with DMF and ether, and dried under reduced pressure to giveBoc-Cys(Npys)-Arg(Pmc)-Met-Phe-Pro-Asn(Trt)-Ala-Pro-Tyr(tBu)-Leu-Alko-resin(630 mg). To this peptide resin was added a mixture ofTFA/H₂O/TIS=95/2.5/2.5 (10 ml), and the mixture was shaken at roomtemperature for 2 hr. The resin was filtered off, and the reactionmixture was concentrated under reduced pressure. The reaction mixturewas ice-cooled and diethyl ether (50 ml) was added. The resultingprecipitate was collected by filtration, washed with ether and driedunder reduced pressure to give crude peptide (217 mg). The obtainedcrude peptide solution was dissolved in a mixture of 20% aqueous aceticacid (7 ml) and acetonitrile (1 ml) and purified by reversed-phase HPLC.

pump: manufactured by Shimadzu; LC-8A

column: YMC ODS-A 3 cmφ×25 cmL, 10 μm

eluate 1: H₂O/0.1% TFA

eluate 2: CH₃CN/0.1% TFA

flow rate: 20 ml/min

detection: UV220 nm

The crude peptide solution was injected to a column equilibrated with15% of eluate 2. Thereafter, the concentration of eluate 2 was raised to37% over 10 min, and thereafter raised at a rate of 0.24% per minute.Fractions containing the object product were collected and freeze driedto give H-Cys(Npys)-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH (53 mg).

mass spectrometry: LC-ESI/MS m/z=1366.1 [M+1]⁺ (Calculated=1366.6)

Step 2. Synthesis of(H-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH)(H-Cys-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH)Disulfide Bond

[That is, synthesis of a compound represented by the formula (5):

wherein the bond between C and C is a disulfide bond.]

H-Cys(Npys)-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH (50 mg) obtained instep 1 and H-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH (i.e., CYTWNQMNL(SEQ ID NO: 4)) (43 mg) synthesized by a known method (e.g.,WO07/063903) were mixed, DMSO (1 mL) was added, and the mixture wasstirred at room temperature for 20 min. The reaction mixture was dilutedwith 0.1% TFA water (5 ml) and purified by reversed-phase HPLC.

pump: manufactured by Shimadzu; LC-8A

column: YMC ODS-A 3 cmφ×25 cmL, 10 μm

eluate 1: H₂O/0.1% TFA

eluate 2: CH₃CN/0.1% TFA

flow rate: 20 ml/min

detection: UV220 nm

The reaction solution was injected to a column equilibrated with 25% ofeluate 2. Thereafter, the concentration of eluate 2 was raised at a rateof 0.25% per minute. Fractions containing the object product werecollected, freeze dried, re-purified by reversed-phase HPLC, and freezedried to give(H-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH)(H-Cys-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH)disulfide bond (i.e., a compound represented by the formula (5), 21 mg).

mass spectrometry: LC-ESI/MS m/z=1191.8 [M+2]²⁺ (Calculated=1191.9)

Example 2 Synthesis of Peptide Consisting of the Following Amino AcidSequence

CRMFPNAPYL (SEQ ID NO: 13)

step 1. Using Fmoc-Leu-Alko-resin (Alko is p-alkoxybenzylalcohol) (338mg, manufactured by Watanabe Chemical; 0.74 mmol/g, 0.25 mmol) as astarting material, and solid phase synthesis as in the method describedin Example 1 was performed twice to giveH-Cys(Trt)-Arg(Pmc)-Met-Phe-Pro-Asn(Trt)-Ala-Pro-Tyr(tBu)-Leu-Alko-resin(1.54 g). To this peptide resin was added a mixture ofTFA/H₂O/TIS=95/2.5/2.5 (15 ml), and the mixture was shaken at roomtemperature for 3 hr. The resin was filtered off, and the reactionmixture was concentrated under reduced pressure. The reaction mixturewas ice-cooled and diethyl ether (50 ml) was added. The resultingprecipitate was collected by filtration, washed with ether and driedunder reduced pressure to give crude peptide (637 mg).

mass spectrometry: LC-ESI/MS m/z=1211.9 [M+1]⁺ (Calculated=1212.5)

step 2. The crude peptide (321 mg) obtained in step 1 was dissolved inTFA (10 ml), and charged, by a pump of HPLC, into a YMC-PACK ODS-A 3cmφ×25 cmL column equilibrated with HPLC (manufactured by Shimadzu;LC6AD) eluate 1=H₂O/0.1% TFA. This state was maintained for about 20 minand, after 20 min, the concentration of eluate 2=CH₃CN/0.1% TFA wasraised to 270. Thereafter, while monitoring the eluate of the objectpeptide by 220 nm UV, the concentration of eluate 2 was raised at a rateof 0.25% per minute and the fractions containing the object product werecollected. The peptide (100 mg) obtained after freeze dry was purifiedagain by reversed-phase under the same conditions, and acetonitrile wasevaporated under reduced pressure and the residue was freeze dried togive the object peptide (CRMFPNAPYL (SEQ ID NO: 13), 37.2 mg).

pump: manufactured by Shimadzu; LC-6A

column: YMC ODS-A 3 cmφ×25 cmL, 10 μm

eluate 1: H₂O/0.1% TFA

eluate 2: CH₃CN/0.1% TFA

flow rate: 20 ml/min

detection: UV220 nm

mass spectrometry: LC-ESI/MS m/z=1212.0 [M+1]⁺ (Calculated=1211.6)

Examples 3-5

By a method similar to that in Example 2, peptides consisting of theamino acid sequence of SEQ ID NO: 16, 18 or 17 were synthesized. Table54 shows the synthesized amount and the results of mass spectrometry.

TABLE 54 mass synthe- spectro- mass amino se- sized metry: spectro- Ex.acid quence amount LC-ESI/ metry: No. sequence No. (mg) MS m/zCalculated 3 CALLPAVPS 16 42  983.8  983.2 L [M + 1]⁺ 4 CRVPGVAPT 18 531012.7 1012.2 L [M + 1H]⁺ 5 CSLGEQQYS 17 31 1113.7 1113.2 V [M + 1]⁺

Experimental Example 1 Time-Course Changes of Trimming of N-TerminalAmino Acid by ERAP1

The peptides of SEQ ID NOs: 13, 16, 18 and 17 synthesized in Examples2-5 were evaluated for the trimming of the N-terminal amino acid byERAP1 (PLoS One November 2008, vol. 3, Issue 11, e3658).

30 μl of ERAP1 (2.0 mg/ml) in PBS buffer solution was added to 258 μl ofTrisHCl buffer. DMSO solution (12.0 μl) of 10 mM each peptide was addedto the aforementioned ERAP1 solution, and the mixture was blended welland stood at room temperature. 1.0, 2.0, 4.0, 8.0 hr later, 50 μl of asample was added to 150 μl of MeOH to terminate the reaction, 25 μl wasinjected into UFLC (analysis conditions shown below), and AUC of theobject peptide was determined. Peptide obtained by trimming waschemically synthesized separately, and analyzed under similar conditionsfree of enzyme. The formation ratio of peptide obtained by trimming wasdetermined based on the obtained AUC.

Analysis Conditions

pump: UFLC manufactured by Shimadzu

column: Shim-pack XR-ODS 3.0 mmi.d.×75 mm

solution: 0.1% TFA H₂O(A)-0.1% TFA CH₃CN(B)

oven temperature: 40° C.

flow rate: 1.0 ml/min

detection wavelength: λ=220 nm

gradient:

-   1. Concentration of SOLUTION B was raised from 1.0% to 70% from 0.0    min to 5.0 min-   2. Concentration of SOLUTION B was raised from 1.0% to 50% from 0.0    min to 5.0 min object peptide:

As for the peptides synthesized in Examples 2-5, the amino acidsequences of the peptides obtained by trimming of N-terminal amino acidby ERAP1 are shown in Table 55.

TABLE 55 peptide used for  peptide obtained  trimming test by trimmingamino  amino  Example acid sequence acid sequence No. sequence No.sequence No. 2 CRMFPNAPYL 13 RMFPNAPYL 2 3 CALLPAVPSL 16 ALLPAVPSL 5 4CRVPGVAPTL 18 RVPGVAPTL 7 5 CSLGEQQYSV 17 SLGEQQYSV 6

Time-course changes in the formation rate of the peptides obtained bytrimming are shown in Table 56 and FIG. 1.

TABLE 56 formation rate (%) Example sequence 1 hr 2 hr 4 hr 8 hr No. No.gradient later later later later 2 13 1 25.5 35.2 46.2 47.6 3 16 1 65.550.6 13.5 0 4 18 1 59.1 57.5 30.1 7.80 5 17 2 77.6 72.8 46.0 7.90

The trimming results strongly suggest that, in any Cys-extended peptides(SEQ ID NOs: 13, 16, 17 and 18), Cys on the extended N-terminal isselectively cleaved by ERAP-1, namely, Cys-extended peptide undergoesappropriate trimming by ERAP-1 without marked dependence on the peptidesequence, and is finally converted to the object cancer antigen peptide(SEQ ID NO: 2, 5, 6 or 7).

Experimental Example 2 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse and HLA-A2402 Transgenic Mouse

The compound represented by the formula (5) synthesized in Example 1 wasevaluated for the CTL induction ability by an in vivo CTL induction testusing HLA-A0201 transgenic mouse and HLA-A2402 transgenic mouse. Thecompound represented by the formula (5):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is RMFPNAPYL (SEQ ID NO: 2) and cancer antigen peptide B isCYTWNQMNL (SEQ ID NO: 4). RMFPNAPYL (SEQ ID NO: 2) is aHLA-A0201-restricted WT1 peptide, and CYTWNQMNL (SEQ ID NO: 4) is aHLA-A24-restricted WT1 peptide.

HLA-A0201 transgenic mouse (C57BL/6CrHLA-A2.1DR1) is a mouse which isdefective in mouse MHC, and expresses chimera HLA of human MHC HLA-A0201and mouse MHC H-2D^(b), and HLA-DRB1*0101. Using this mouse, HLA-A02positive peptide capable of inducing CTL in human can be selected (Eur JImmunol. 2004; 34: 3060-9). On the other hand, HLA-A2402 transgenicmouse (C57BL/6CrHLA-A2402/K^(b)) is a mouse that expresses chimera HLAof human MHC HLA-A2402 and mouse MHC H-2K^(b). Using this mouse, HLA-A24positive peptide capable of inducing CTL in human can be selected (Int JCancer. 2002; 100: 565-70).

Whether the administration of a compound represented by the formula (5)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2, 4) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2, 4), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (5).

Specifically, a compound represented by the formula (5) was dissolved indimethyl sulfoxide (DMSO) at 40 mg/mL, further diluted with water forinjection to 5 mg/mL, and emulsified by mixing with an equal amount ofincomplete Freund's adjuvant (IFA). The emulsified compound wasintradermally administered to 4 sites at the base of tail of a mouse at250 μg/site. One week later, the mouse was euthanized with CO₂ gas, thespleen was isolated, and splenocytes were prepared. IFNγ ELISPOT assaykit was used for the measurement of IFNγ production. On the previous dayof splenocyte preparation, an ELISPOT plate was treated with ananti-mouse IFNγ antibody, and blocked with RPMI1640 medium containing10% FBS the next day. The prepared HLA-A0201 transgenic mouse-derivedsplenocytes were plated at 0.15×10⁶ cells/well, and HLA-A2402 transgenicmouse-derived splenocytes were plated at 1×10⁶ cells/well, on theblocked ELISPOT plate. Peptide (SEQ ID NO: 2, 4) was dissolved in DMSOat 40 mg/mL, and further diluted with RPMI1640 medium containing 10% FBSto 40 μg/mL. The diluted peptide (SEQ ID NO: 2) was added to theHLA-A0201 transgenic mouse-derived splenocytes at a final concentrationof 10 μg/mL. In addition, the diluted peptide (SEQ ID NO: 4) was addedto the HLA-A2402 transgenic mouse-derived splenocytes at a finalconcentration of 10 μg/mL. The splenocytes added with the peptide werecultivated for 20 hr at 37° C., 5% CO₂, whereby peptide re-stimulationin vitro was performed. After culture, the supernatant was removed, andthe ELISPOT plate was allowed to develop color according to the attachedprotocol. The number of spots that developed color was measured byImmunoSpot Analyzer (manufactured by C.T.L.).

The results of IFNγ ELISPOT assay using HLA-A0201 transgenic mouse areshown in FIG. 2, and the results of IFNγ ELISPOT assay using HLA-A2402transgenic mouse are shown in FIG. 3.

In each Figure, the vertical axis shows the number of cells that reactedin the plated cells. In FIG. 2, the black bar and the white bar show theresults of culture of HLA-A0201 transgenic mouse-derived splenocytes inthe presence or absence of the object peptide represented by SEQ ID NO:2, and in FIG. 3, the black bar and the white bar show the results ofculture of HLA-A2402 transgenic mouse-derived splenocytes in thepresence or absence of the object peptide represented by SEQ ID NO: 4.That is, the difference in the values of the black bar and the white barshow the number of the object, each peptide-specific CTL induced in themouse in vivo by the administration of a compound represented by theformula (5).

In each Figure, the value of the white bar is not detected. This meansthat the splenocytes of respective transgenic mice did not react at allin the absence of the object peptide. As a result of this test, IFNγproduction specific to the object peptide shown by SEQ ID NO: 2 wasdetected in the HLA-A0201 transgenic mouse-derived splenocytes, and IFNγproduction specific to the object peptide shown by SEQ ID NO: 4 wasdetected in the HLA-A2402 transgenic mouse-derived splenocytes.

From the above, it was clarified that a compound represented by theformula (5) can induce CTL specific to the peptide shown by SEQ ID NO: 2and CTL specific to the peptide shown by SEQ ID NO: 4. It was stronglysuggested that the compound represented by the formula (5) undergoescleavage of disulfide bond and appropriate trimming by ERAP-1 in mousein vivo and is in fact processed into the peptides shown by SEQ ID NO: 2and SEQ ID NO: 4.

That is, it was clarified that a compound represented by the formula(5), which is one embodiment of the compound of the present invention,is a conjugate wherein different two kinds of WT1 peptides form acomposite via the disulfide bond shown in the formula (1), and is a WT1cancer antigen peptide conjugate vaccine that in fact can inducedifferent two kinds of CTLs in vivo.

Reference Examples 1-7

By a method similar to that in Example 2, respective peptides consistingof the amino acid sequences of SEQ ID NOs: 22, 24, 23, 2, 4, 6 and 5were synthesized. Table 57 shows the results of mass spectrometry. SinceSEQ ID NOs: 22, 24, 23, 2, 4, 6 and 5 are not the compound of thepresent invention, they are described as Reference Examples.

TABLE 57 mass mass spectro- spectro- Ref. se- metry: metry: Ex.amino acid quence LC-ESI/ Calcu- No. sequence No. MS m/z lated 1CNKRYFKLSHLQMHSRK 22 1089.1 1089.3 [M + 2H]⁺ 2 CNKRYFKLSHLQMHSRKH 24 825.1  824.8 TG [M + 3H]⁺ 3 CNKRYFKLSHLQMHSRKH 23  772.4  772.2 [M +3H]⁺ 4 RMFPNAPYL  2 1109.0 1109.3 [M + H]⁺ 5 CYTWNQMNL  4 1172.9 1173.4[M + H]⁺ 6 SLGEQQYSV  6 1010.9 1011.1 [M + H]⁺ 7 ALLPAVPSL  5  881.0 881.1 [M + H]⁺

Examples 6-9

By a method similar to that in Example 1, respective compounds(conjugates) represented by the formulas (3), (6), (7) and (8) weresynthesized. Table 58 shows the results of mass spectrometry. (In eachformula, the bond between C and C is a disulfide bond.)

TABLE 58 mass mass spectrometry: spectro- Ex. formula LC-ESI/ metry: No.structural formula No. MS m/z Calculated 6

(3) 1162.3 [M + 2H]⁺ 1162.0 7

(6) 1228.0 [M + 3H]⁺ 1227.6 8

(7) 705.8 [M + 5H]⁺ 705.3 9

(8) 1129.8 [M + 3H]⁺ 1129.2

Experimental Example 3 Measurement of Solubility Step 1. Preparation ofIsotonic Buffer

1. 75% aqueous solution of disodium hydrogen phosphate and 5.53% aqueoussolution of citric acid were mixed, and respective buffers (pH 6.0 and7.4) were prepared.

Step 2. Preparation of Test Solution

About 1 mg of a test product was measured, an isotonic buffer (0.5 mL)was added, and this was used as a test solution. The prepared testsolution was shaken at room temperature for 90 min (shaking conditions:RECIPRO SHAKER SR-1N manufactured by TAITEC, Speed=8), centrifuged(15000 rpm, 5 min, room temperature), and the supernatant aftercentrifugation was used as a test solution.

Step 3. Preparation of Standard Solution

About 1 mg of the test product was accurately measured, dissolved in0.1% TFA water/acetonitrile=l/l, made the total amount 10 mL, and thiswas used as a standard solution of the test product.

Step 4. Measurement of Concentration of Test Product

The standard solution of the test product and the test solution wereanalyzed by HPLC (analysis conditions described in Table 59), and thesolubility of the test product was calculated from the peak area ratioof the standard solution.

HPLC measurement conditions

column: ChemcoPack Quicksorb (4.6 mmφ×150 mm, 5 μm)

manufactured by Chemco Scientific Co., Ltd.

mobile phase: SOLUTION A; 0.1% TFA water, SOLUTION B; 0.1% TFAacetonitrile solution

column temperature: room temperature

flow rate: 1 mL/min

detection wavelength: UV 254 nm, 230 nm (2 wavelength detection)

sample injection volume: 10 μL

TABLE 59 gradient analysis conditions time (min) SOLUTION A (%) SOLUTIONB (%) 0.00 80 20 10.00 0 100 15.00 0 100 15.01 80 20 25.00 80 20 25.01STOP

The peptides synthesized in Reference Examples 1-2 and 4-7 and thecompounds (conjugates) synthesized in Examples 1, 7 and 9 were subjectedto the above-mentioned solubility measurement. Each solubility is shownin Table 60.

TABLE 60 SEQ ID amino acid NO: Reference sequence or or Example No. orstructural formula pH 6.0 pH 7.4 Example No. formula No. (mg/mL) (mg/mL)Reference SLGEQQYSV SEQ ID >1.0 >1.0 Example 6 NO: 6 Reference ALLPAVPSLSEQ ID >1.0 >1.0 Example 7 NO: 5 Reference CNKRYFKLSHLQMHSRKHTGSEQ ID >1.0 0.556 Example 2 NO: 24 Reference CNKRYFKLSHLQMHSRKSEQ ID >1.0 0.931 Example 1 NO: 22 Example 7

formula (6) >1.0 0.279 Example 9

formula (8) >1.0 0.789 Reference RMFPNAPYL SEQ ID >1.0 >1.0 Example 4NO: 2 Reference CYTWNQMNL SEQ ID 0.106 0.167 Example 5 NO: 4 Example 1

formula (5) 0.511 0.200

Experimental Example 4 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse

The compound represented by the formula (3) synthesized in Example 6 wasevaluated for the CTL induction ability by an in vivo CTL induction testusing HLA-A0201 transgenic mouse. The compound represented by theformula (3):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is RMFPNAPYL (SEQ ID NO: 2) and cancer antigen peptide B isSLGEQQYSV (SEQ ID NO: 6). RMFPNAPYL (SEQ ID NO: 2) and SLGEQQYSV (SEQ IDNO: 6) are a HLA-A0201-restricted WT1 peptides.

The HLA-A0201 transgenic mouse is as described in Experimental Example2.

Whether the administration of a compound represented by the formula (3)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2, 6) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2, 6), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (3).

Specifically, a compound represented by the formula (3) was dissolved inwater for injection at 10 mg/mL, and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified compoundwas intradermally administered to 2 sites at the base of tail of a mouseat 500 μg/site. One week later, the mouse was euthanized with CO₂ gas,the spleen was isolated, and splenocytes were prepared. IFNγ ELISPOTassay kit was used for the measurement of IFNγ production. On theprevious day of splenocyte preparation, an ELISPOT plate was treatedwith an anti-mouse IFNγ antibody, and blocked with RPMI1640 mediumcontaining 10% FBS the next day. The prepared HLA-A0201 transgenicmouse-derived splenocytes were plated at 0.75×10⁶ cells/well on theblocked ELISPOT plate. Peptide (SEQ ID NO: 2, 6) was dissolved in DMSOat 40 mg/mL, and further diluted with RPMI1640 medium containing 10% FBSto 40 μg/mL. The diluted peptide (SEQ ID NO: 2, 6) was added to theHLA-A0201 transgenic mouse-derived splenocytes at a final concentrationof 10 μg/mL. The splenocytes added with the peptide were cultured for 20hr at 37° C., 5% CO₂, whereby peptide re-stimulation in vitro wasperformed. After culture, the supernatant was removed, and the ELISPOTplate was allowed to develop color according to the attached protocol.The number of spots that developed color was measured by ImmunoSpotAnalyzer (manufactured by C.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 4.

In FIG. 4, the vertical axis shows the number of cells that reacted inthe plated cells. In FIG. 4, the black bar and the shaded bar show theresults of culture of HLA-A0201 transgenic mouse-derived splenocyteswhile being pulsed with each peptide shown by SEQ ID NO: 2, 6, and thewhite bar show the results of culture without pulsing. That is, thedifference in the values of the black or shaded bar and the white barshows the number of peptide-specific CTL, and that the administration ofa compound represented by the formula (3) resulted in the induction ofCTL specific to each peptide shown by SEQ ID NOs: 2, 6 in vivo in themouse. In FIG. 4, the value of the white bar is not detected. This meansthat the splenocytes of HLA-A0201 transgenic mice did not react at allin the absence of pulsing with the object peptide. As a result of thistest, IFNγ production specific to the peptide shown by SEQ ID NO: 2, 6was detected in the HLA-A0201 transgenic mouse-derived splenocytes.

From the above, it was clarified that a compound represented by theformula (3) can induce CTL specific to the peptide shown by SEQ ID NO:2, 6. It was strongly suggested that the compound represented by theformula (3) undergoes cleavage of disulfide bond and appropriatetrimming by ERAP-1 in mouse in vivo and is in fact processed into thepeptides shown by SEQ ID NO: 2 and 6. That is, it was clarified that acompound represented by the formula (3), which is one embodiment of thecompound of the present invention, is a conjugate wherein different twokinds of peptides form a composite via the disulfide bond shown in theformula (1), and is a WT1 cancer antigen peptide conjugate vaccine thatin fact can induce different two kinds of CTLs in vivo.

Experimental Example 5 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse

The compound represented by the formula (6) synthesized in Example 7 wasevaluated for the CTL induction ability by an in vivo CTL induction testusing HLA-A0201 transgenic mouse. The compound represented by theformula (6):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is RMFPNAPYL (SEQ ID NO: 2) and cancer antigen peptide C isCNKRYFKLSHLQMHSRKHTG (SEQ ID NO: 24). RMFPNAPYL (SEQ ID NO: 2) is aHLA-A0201-restricted WT1 peptide, and CNKRYFKLSHLQMHSRKHTG (SEQ ID NO:24) is an MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Example2. Using this mouse, HLA-A02 positive peptide capable of inducing CTL inhuman can be selected, as well as the CTL induction enhancing activityof helper peptide capable of inducing helper T cell by binding to humanHLA-DRB1*0101 can be evaluated.

Whether the administration of a compound represented by the formula (6)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) and cells reactive with helper peptide (SEQ ID NO: 24) was judgedbased on the measurement of IFNγ production by re-stimulation, with thepeptide (SEQ ID NO: 2, 24), of the splenocyte derived from theabove-mentioned mouse administered with a compound represented by theformula (6). In addition, the splenocytes derived from theabove-mentioned mouse administered with a compound represented by theformula (6) and the splenocytes derived from the above-mentioned mouseadministered with a compound represented by SEQ ID NO: 2 werere-stimulated with the peptide (SEQ ID NO: 2), and the IFNγ-producingcell numbers were compared.

Specifically, a peptide represented by SEQ ID NO: 2 was dissolved inwater for injection at 6 mg/mL, and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified peptide wasintradermally administered to 2 sites at the base of tail of a mouse at150 μg/site. A compound represented by the formula (6) was dissolved inwater for injection (19.8 mg/mL), and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified compoundwas intradermally administered to 2 sites at the base of tail of a mouseat 495 fig/site. The mole number of the peptide of SEQ ID NO: 2contained in the dose of the compound represented by the formula (6) perone mouse was adjusted to be equal to that of the peptide of SEQ ID NO:2 contained in the dose per mouse. One week later, the mouse waseuthanized with CO₂ gas, the spleen was isolated, and splenocytes wereprepared. IFNγ ELISPOT assay kit was used for the measurement of IFNγproduction. On the previous day of splenocyte preparation, an ELISPOTplate was treated with an anti-mouse IFNγ antibody, and blocked withRPMI1640 medium containing 10% FBS the next day. The prepared HLA-A0201transgenic mouse-derived splenocytes were plated at 0.25×10⁶ iscells/well or 0.5×10⁶ cells/well on the blocked ELISPOT plate. Peptide(SEQ ID NO: 2, 24) was dissolved in DMSO at 40 mg/mL, and furtherdiluted with RPMI1640 medium containing 10% FBS to 40 μg/mL. The dilutedpeptide (SEQ ID NO: 2, 24) was added to the HLA-A0201 transgenicmouse-derived splenocytes at a final concentration of 10 μg/mL. Thesplenocytes added with the peptide were cultured for 20 hr at 37° C., 5%CO₂, whereby peptide re-stimulation in vitro was performed. Afterculture, the supernatant was removed, and the ELISPOT plate was allowedto develop color according to the attached protocol. The number of spotsthat developed color was measured by ImmunoSpot Analyzer (manufacturedby C.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIGS. 5 and 6.

In FIGS. 5 and 6, the vertical axis shows the number of cells thatreacted in the plated cells, and the horizontal axis shows compound orpeptide administered to the mouse. In FIG. 5, the black bar shows theresults of culture of HLA-A0201 transgenic mouse-derived splenocyteswhile being pulsed with the peptide shown by SEQ ID NO: 2, and the whitebar show the results of culture without pulsing. That is, the differencein the values of the black bar and the white bar shows the number ofpeptide-specific CTL, and that the administration of the peptide shownby SEQ ID NO: 2 or a compound represented by the formula (6) resulted inthe induction of CTL specific to the peptide shown by SEQ ID NO: 2 invivo in the mouse. In FIG. 5, the value of the white bar is notdetected. This means that the splenocytes of HLA-A0201 transgenic micedid not react at all in the absence of pulsing with the object peptide.As a result of this test, IFNγ production specific to the peptide shownby SEQ ID NO: 2 was detected in the HLA-A0201 transgenic mouse-derivedsplenocytes. Moreover, in FIG. 5, the number of IFNγ-producing cellsspecific to the peptide shown by SEQ ID NO: 2, which were induced by theadministration of a compound represented by the formula (6), was higherthan that of the peptide-specific IFNγ-producing cells induced by theadministration of the peptide shown by SEQ ID NO: 2.

In FIG. 6, furthermore, the black bar shows the results of culture ofHLA-A0201 transgenic mouse-derived splenocytes while being pulsed withpeptide shown by SEQ ID NO: 24, and the white bar show the results ofculture without pulsing. That is, the difference in the values of theblack bar and the white bar shows the number of peptide-reactive cells,and that the administration of a compound represented by the formula (6)resulted in the induction of cells reactive with the helper peptideshown by SEQ ID NO: 24 in vivo in the mouse, and administration of acompound represented by SEQ ID NO: 2 did not induce cells reactive withthe peptide shown by SEQ ID NO: 24 in vivo in the mouse. In FIG. 6, thevalue of the white bar is not detected. This means that the splenocytesof HLA-A0201 transgenic mice did not react at all in the absence ofpulsing with the object peptide.

From the above, it was clarified that a compound represented by theformula (6) can induce CTL specific to the peptide shown by SEQ ID NO: 2and cells reactive with the helper peptide shown by SEQ ID NO: 24. Itwas strongly suggested that the compound represented by the formula (6)undergoes cleavage of disulfide bond and appropriate trimming by ERAP-1in mouse in vivo and is in fact processed into the peptides shown by SEQID NO: 2 and 24. It was assumed that the induction of the cell reactivewith the helper peptide shown by SEQ ID NO: 24 produced from a compoundrepresented by the formula (6) enhanced induction of CTL specific to thepeptide shown by SEQ ID NO: 2, and many IFNγ-producing cells specific tothe peptide shown by SEQ ID NO: 2 were found, as compared to theadministration of the peptide shown by SEQ ID NO: 2.

That is, it was clarified that a compound represented by the formula(6), which is one embodiment of the compound of the present invention,is a conjugate wherein two different kinds of peptides form a compositevia the disulfide bond shown in the formula (1), and is a WT1 cancerantigen peptide conjugate vaccine that in fact can induce CTLs andhelper peptide reactive cells in vivo.

Experimental Example 6 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse

The compound represented by the formula (8) synthesized in Example 9 wasevaluated for the CTL induction ability by an in vivo CTL induction testusing HLA-A0201 transgenic mouse. The compound represented by theformula (8):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is RMFPNAPYL (SEQ ID NO: 2) and cancer antigen peptide C isCNKRYFKLSHLQMHSRK (SEQ ID NO: 22). RMFPNAPYL (SEQ ID NO: 2) is aHLA-A0201-restricted WT1 peptide, and CNKRYFKLSHLQMHSRK (SEQ ID NO: 22)is an MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (8)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) and cells reactive with helper peptide (SEQ ID NO: 22) reactivecell was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2, 22), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (8). In addition, the splenocytes derivedfrom the above-mentioned mouse administered with a compound representedby the formula (8) and the splenocytes derived from the above-mentionedmouse administered with a compound represented by SEQ ID NO: 2 werere-stimulated with the peptide (SEQ ID NO: 2), and the IFNγ-producingcell numbers were compared.

Specifically, a peptide represented by SEQ ID NO: 2 was dissolved inwater for injection at 6 mg/mL, and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified peptide wasintradermally administered to 2 sites at the base of tail of a mouse at150 μg/site. A compound represented by the formula (8) was dissolved inwater for injection (18 mg/mL), and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified compoundwas intradermally administered to 2 sites at the base of tail of a mouseat 450 lag/site. The mole number of the peptide of SEQ ID NO: 2contained in the dose of the compound represented by the formula (8) perone mouse was adjusted to be equal to that of the peptide of SEQ ID NO:2 contained in the dose per mouse. One week later, the mouse waseuthanized with CO₂ gas, the spleen was isolated, and splenocytes wereprepared. IFNγ ELISPOT assay kit was used for the measurement of IFNγproduction. On the previous day of splenocyte preparation, an ELISPOTplate was treated with an anti-mouse IFNγ antibody, and blocked withRPMI1640 medium containing 10% FBS the next day. The prepared HLA-A0201transgenic mouse-derived splenocytes were plated at 0.25×10⁶ cells/wellor 0.5×10⁶ cells/well on the blocked ELISPOT plate. Peptide (SEQ ID NO:2, 22) was dissolved in DMSO at 40 mg/mL, and further diluted withRPMI1640 medium containing 10% FBS to 40 μg/mL. The diluted peptide (SEQID NO: 2, 22) was added to the HLA-A0201 transgenic mouse-derivedsplenocytes at a final concentration of 10 μg/mL. The splenocytes addedwith the peptide were cultured for 20 hr at 37° C., 5% CO₂, wherebypeptide re-stimulation in vitro was performed. After culture, thesupernatant was removed, and the ELISPOT plate was allowed to developcolor according to the attached protocol. The number of spots thatdeveloped color was measured by ImmunoSpot Analyzer (manufactured byC.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIGS. 7 and 8.

In FIGS. 7 and 8, the vertical axis shows the number of cells thatreacted in the plated cells, and the horizontal axis shows compound orpeptide administered to the mouse. In FIG. 7, the black bar shows theresults of culture of HLA-A0201 transgenic mouse-derived splenocyteswhile being pulsed with the peptide shown by SEQ ID NO: 2, and the whitebar shows the results of culture without pulsing. That is, thedifference in the values of the black bar and the white bar shows thenumber of peptide-specific CTL, and that the administration of thepeptide shown by SEQ ID NO: 2 or a compound represented by the formula(8) resulted in the induction of CTL specific to the peptide shown bySEQ ID NO: 2 in vivo in the mouse. In FIG. 7, the value of the white baris not detected. This means that the splenocytes of HLA-A0201 transgenicmice did not react at all in the absence of pulsing with the objectpeptide. As a result of this test, IFNγ production specific to thepeptide shown by SEQ ID NO: 2 was detected in the HLA-A0201 transgenicmouse-derived splenocytes. Moreover, in FIG. 7, the number ofIFNγ-producing cells specific to the peptide shown by SEQ ID NO: 2,which were induced by the administration of a compound represented bythe formula (8), was higher than that of the peptide-specificIFNγ-producing cells induced by the administration of the peptide shownby SEQ ID NO: 2.

In FIG. 8, furthermore, the black bar shows the results of culture ofHLA-A0201 transgenic mouse-derived splenocytes is while being pulsedwith peptide shown by SEQ ID NO: 22, and the white bar shows the resultsof culture without pulsing. That is, the difference in the values of theblack bar and the white bar shows the number of peptide-reactive cells,and that the administration of a compound represented by the formula (8)resulted in the induction of cells reactive with the helper peptideshown by SEQ ID NO: 22 in vivo in the mouse, and administration of apeptide represented by SEQ ID NO: 2 did not induce cells reactive withthe peptide shown by SEQ ID NO: 22 in vivo in the mouse. In FIG. 8, thevalue of the white bar is not detected. This means that the splenocytesof HLA-A0201 transgenic mice did not react at all in the absence ofpulsing with the object peptide.

From the above, it was clarified that a compound represented by theformula (8) can induce CTL specific to the peptide shown by SEQ ID NO: 2and cells reactive with the helper peptide shown by SEQ ID NO: 22. Itwas strongly suggested that the compound represented by the formula (8)undergoes cleavage of disulfide bond and appropriate trimming by ERAP-1in mouse in vivo and is in fact processed into the peptides shown by SEQID NO: 2 and 22. It was assumed that the induction of the cell reactivewith the helper peptide shown by SEQ ID NO: 22 produced from a compoundrepresented by the formula (8) enhanced induction of CTL specific to thepeptide shown by SEQ ID NO: 2, and many IFNγ-producing cells specific tothe peptide shown by SEQ ID NO: 2 were found, as compared to theadministration of the compound shown by SEQ ID NO: 2.

That is, it was clarified that a compound represented by the formula(8), which is one embodiment of the compound of the present invention,is a conjugate wherein two different kinds of peptides form a compositevia the disulfide bond shown in the formula (1), and is a WT1 cancerantigen peptide conjugate vaccine that in fact can induce CTLs andhelper peptide reactive cells in vivo.

Example 10

By a method similar to that in Example 1, respective compounds(conjugates) represented by the formula (9) were synthesized. Table 61shows the results of mass spectrometry. (In each formula, the bondbetween C and C is a disulfide bond.)

TABLE 61 mass spectrometry: mass Ex. formula LC-ESI/MS spectrometry: No.structural formula No. m/Z Calculated 10

9 1151.9 [M + 3H]³⁺ 1152.0

Reference Example 8-9

By a method similar to that in Example 2, peptides consisting of theamino acid sequences shown by SEQ ID NOs: 238-239 were synthesized. Theresults of mass spectrometry are shown in Table 62. Since the peptidesdescribed in the Table are not the compound of the present invention,they are indicated as Reference Examples.

TABLE 62 mass spectro- metry: mass Ref. SEQ  LC- spectro- Ex. amino acidID ESI/MS metry: No. sequence NO: m/Z Calculated 8 RMFPNAPYLCYTWNQMNL238 1132.2 1132.3 [M + 2H]²⁺ 9 CYTWNQMNLRMFPNAPYL 239 1133.0 1132.3 [M +2H]²⁺

Reference Examples 10-11

By a method similar to that in Example 2, peptides consisting of theamino acid sequences shown by SEQ ID NOs: 240-241 were synthesized. Theresults of mass spectrometry are shown in Table 63. Since the peptidesdescribed in the Table are not the compound of the present invention,they are indicated as Reference Examples.

TABLE 63 mass mass spectro- spectro- Ref. SEQ metry: metry: Ex. IDLC-ESI/ Calcu- No. amino acid sequence NO: MS m/ lated 10RMFPNAPYLGGGGGGCYTWNQMNL 240 1303.7 1303.5 [M + 2H]²⁺ 11CYTWNQMNGGGGGGRMFPNAPYL 241 1303.0 1303.5 [M + 2H]²⁺

The peptides shown in Table 63 were synthesized by referring to thenon-patent document, Cancer Science January 2012, Vol. 103, no. 1,150-153.

Experimental Example 7 Stability Test of Conjugate and Cocktail VaccineStep 1

Conjugate (formula No.: (6)) (2.4 mg) was dissolved in 120 μL of waterfor injection and preserved under shading at room temperature.

Step 2

As a cocktail vaccine, the peptide shown by SEQ ID NO: 2 (1.1 mg) wasdissolved in 180 μL of water for injection, 123 μL thereof was used todissolve the peptide shown by SEQ ID NO: 24 (1.3 mg), and the solutionwas preserved under shading at room temperature.

Step 3

The solutions (2.5 μL) obtained in step 1 and step 2 were diluted withwater for injection (50 μL) and subjected to HPLC analysis (analysisconditions are shown below), and the content percentage of the conjugateand peptide in the aqueous solution were measured with the area valueimmediately after the start of the preservation as 100%. The contentpercentage of the conjugate is shown in Table 64, and that of eachpeptide in the cocktail vaccine is shown in Table 65.

analysis conditions

pump: UFLC manufactured by Shimadzu

column: Kinetex 2.6u C18 100A 3.0 mm i.d.×75 mm

mobile phase: SOLUTION A; 0.1% TFA water, SOLUTION B; 0.1% TFAacetonitrile solution

column temperature: 40° C.

flow rate: 1 mL/min

detection wavelength: UV 220, 254 nm (2 wavelengths detection)

sample injection volume: 10 μL

TABLE 64 elapsed formula No. (6) content time percentage (%) 1 day 107 2weeks 96

TABLE 65 elapsed SEQ ID NO: 2 content SEQ ID NO: 24 content timepercentage (%) percentage (%) 1 day 97 65 1 week 99 9 2 weeks 94 6

In Experimental Example 7, the conjugate represented by formula No. (6)contained 96% of the compound represented by the formula (6) at the timepoint of 2 weeks from the solution preparation. In contrast, in a mixedsolution of SEQ ID NO: 2 and SEQ ID NO: 24, which is a cocktail vaccine,the content percentage of SEQ ID NO: 24 decreased to 65% at the timepoint of 1 day elapse, and to 6% 2 weeks later. These results show thatthe conjugate preserved in the form of an aqueous solution is wasstabler than cocktail vaccine preserved under the same conditions.

Experimental Example 8 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse

The compound represented by the formula (7) synthesized in Example 8 wasevaluated for the CTL induction ability by an in vivo CTL induction testusing HLA-A0201 transgenic mouse. The compound represented by theformula (7):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is RMFPNAPYL (SEQ ID NO: 2) and cancer antigen peptide C isCNKRYFKLSHLQMHSRKH (SEQ ID NO: 23). RMFPNAPYL (SEQ ID NO: 2) is aHLA-A0201-restricted WT1 peptide, and CNKRYFKLSHLQMHSRKH (SEQ ID NO: 23)is an MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (7)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) and cells reactive with helper peptide (SEQ ID NO: 23) was judgedbased on the measurement of IFNγ production by re-stimulation, with thepeptide (SEQ ID NO: 2, 23), of the splenocyte derived from theabove-mentioned mouse administered with a compound represented by theformula (7). In addition, the splenocytes derived from theabove-mentioned mouse administered with a compound represented by theformula (7) and the splenocytes derived from the above-mentioned mouseadministered with a compound represented by SEQ ID NO: 2 werere-stimulated with the peptide (SEQ ID NO: 2), and the IFNγ-producingcell numbers were compared.

Specifically, a peptide represented by SEQ ID NO: 2 was dissolved inwater for injection at 6 mg/mL, and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified peptide wasintradermally administered to 2 sites at the base of tail of a mouse at150 lag/site. A compound represented by the formula (7) was dissolved inwater for injection (19 mg/mL), and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified compoundwas intradermally administered to 2 sites at the base of tail of a mouseat 475 μg/site. The mole number of the peptide of SEQ ID NO: 2 containedin the dose of the compound represented by the formula (7) per one mousewas adjusted to be equal to that of the peptide of SEQ ID NO: 2contained in the dose per mouse. One week later, the mouse waseuthanized with CO₂ gas, the spleen was isolated, and splenocytes wereprepared. IFNγ ELISPOT assay kit was used for the measurement of IFNγproduction. On the previous day of splenocyte preparation, an ELISPOTplate was treated with an anti-mouse IFNγ antibody, and blocked withRPMI1640 medium containing 10% FBS the next day. The prepared HLA-A0201transgenic mouse-derived splenocytes were plated at 0.25×10⁶ cells/wellor 0.5×10⁶ cells/well on the blocked ELISPOT plate. Peptide (SEQ ID NO:2, 23) was dissolved in DMSO at 40 mg/mL, and further diluted withRPMI1640 medium containing 10% FBS to 40 μg/mL. The diluted peptide (SEQID NO: 2, 23) was added to the HLA-A0201 transgenic mouse-derivedsplenocytes at a final concentration of 10 μg/mL. The splenocytes addedwith the peptide were cultured for 17 hr at 37° C., 5% CO₂, wherebypeptide re-stimulation in vitro was performed. After culture, thesupernatant was removed, and the ELISPOT plate was allowed to developcolor according to the attached protocol. The number of spots thatdeveloped color was measured by ImmunoSpot Analyzer (manufactured byC.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIGS. 9 and 10. In FIGS. 9 and 10, the vertical axis showsthe number of cells that reacted in the plated cells, and the horizontalaxis shows compound or peptide administered to the mouse. In FIG. 9, theblack bar shows the results of culture of HLA-A0201 transgenicmouse-derived splenocytes while being pulsed with the peptide shown bySEQ ID NO: 2, and the white bar shows the results of culture withoutpulsing. That is, the difference in the values of the black bar and thewhite bar shows the number of peptide-specific CTL, and that theadministration of the peptide shown by SEQ ID NO: 2 or a compoundrepresented by the formula (7) resulted in the induction of CTL specificto the peptide shown by SEQ ID NO: 2 in vivo in the mouse. In FIG. 9,the value of the white bar is not detected. This means that thesplenocytes of HLA-A0201 transgenic mice did not react at all in theabsence of pulsing with the object peptide. As a result of this test,IFNγ production specific to the peptide shown by SEQ ID NO: 2 wasdetected in the HLA-A0201 transgenic mouse-derived splenocytes.Moreover, in FIG. 9, the number of IFNγ-producing cells specific to thepeptide shown by SEQ ID NO: 2, which were induced by the administrationof a compound represented by the formula (7), was higher than that ofthe peptide-specific IFNγ-producing cells induced by the administrationof the peptide shown by SEQ ID NO: 2.

In FIG. 10, furthermore, the black bar shows the results of culture ofHLA-A0201 transgenic mouse-derived splenocytes while being pulsed withpeptide shown by SEQ ID NO: 23, and the white bar shows the results ofculture without pulsing. That is, the difference in the values of theblack bar and the white bar shows the number of peptide-reactive cells,and that the administration of a compound represented by the formula (7)resulted in the induction of cells reactive with the helper peptideshown by SEQ ID NO: 23 in vivo in the mouse, and administration of apeptide represented by SEQ ID NO: 2 did not induce cells reactive withthe peptide shown by SEQ ID NO: 23 in vivo in the mouse. In FIG. 10, thevalue of the white bar is not detected. This means that the splenocytesof HLA-A0201 transgenic mice did not react at all in the absence ofpulsing with the object peptide.

From the above, it was clarified that a compound represented by theformula (7) can induce CTL specific to the peptide shown by SEQ ID NO: 2and cells reactive with the helper peptide shown by SEQ ID NO: 23. Itwas strongly suggested that the compound represented by the formula (7)undergoes cleavage of disulfide bond and appropriate trimming by ERAP-1in mouse in vivo and is in fact processed into the peptides shown by SEQID NO: 2 and 23. It was assumed that the induction of the cell reactivewith the helper peptide shown by SEQ ID NO: 23 produced from a compoundrepresented by the formula (7) enhanced induction of CTL specific to thepeptide shown by SEQ ID NO: 2, and many IFNγ-producing cells specific tothe peptide shown by SEQ ID NO: 2 were found, as compared to theadministration of the compound shown by SEQ ID NO: 2.

That is, it was clarified that a compound represented by the formula(7), which is one embodiment of the compound of the present invention,is a conjugate wherein two different kinds of peptides form a compositevia the disulfide bond shown in the formula (1), and is a WT1 cancerantigen peptide conjugate vaccine that in fact can induce CTLs andhelper peptide reactive cells in vivo.

Experimental Example 9 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse

The compound represented by the formula (9) synthesized in Example 10was evaluated for the CTL induction ability by an in vivo CTL inductiontest using HLA-A0201 transgenic mouse. The compound represented by theformula (9):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is ALLPAVPSL (SEQ ID NO: 5) and cancer antigen peptide C isCNKRYFKLSHLQMHSRKHG (SEQ ID NO: 24). ALLPAVPSL (SEQ ID NO: 5) is aHLA-A0201 and HLA-A2402-restricted WT1 peptide, and CNKRYFKLSHLQMHSRKHG(SEQ ID NO: 24) is an MHC class II-restricted WT1 peptide (namely,helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (9)results in the induction of CTL specific to the object peptide (SEQ IDNO: 5) and cells reactive with helper peptide (SEQ ID NO: 24) was judgedbased on the measurement of IFNγ production by re-stimulation, with thepeptide (SEQ ID NO: 5, 24), of the splenocyte derived from theabove-mentioned mouse administered with a compound represented by theformula (9). In addition, the splenocytes derived from theabove-mentioned mouse administered with a compound represented by theformula (9) and the splenocytes derived from the above-mentioned mouseadministered with a compound represented by SEQ ID NO: 5 werere-stimulated with the peptide (SEQ ID NO: 5), and the IFNγ-producingcell numbers were compared.

Specifically, a peptide represented by SEQ ID NO: 5 was dissolved inwater for injection at 6 mg/mL, and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified peptide wasintradermally administered to 2 sites at the base of tail of a mouse at150 μg/site. A compound represented by the formula (9) was dissolved inwater for injection (23.6 mg/mL), and emulsified by mixing with an equalamount of incomplete Freund's adjuvant (IFA). The emulsified compoundwas intradermally administered to 2 sites at the base of tail of a mouseat 590 μg/site. The mole number of the peptide of SEQ ID NO: 5 containedin the dose of the compound represented by the formula (9) per one mousewas adjusted to be equal to that of the peptide of SEQ ID NO: 5contained in the dose per mouse. One week later, the mouse waseuthanized with CO₂ gas, the spleen was isolated, and splenocytes wereprepared. IFNγ ELISPOT assay kit was used for the measurement of IFNγproduction. On the previous day of splenocyte preparation, an ELISPOTplate was treated with an anti-mouse IFNγ antibody, and blocked withRPMI1640 medium containing 10% FBS the next day. The prepared HLA-A0201transgenic mouse-derived splenocytes were plated at 0.25×10⁶ cells/wellor 0.75×10⁶ cells/well on the blocked ELISPOT plate. Peptide (SEQ ID NO:5, 24) was dissolved in DMSO at 40 mg/mL, and further diluted withRPMI1640 medium containing 10% FBS to 40 μg/mL. The diluted peptide (SEQID NO: 5, 24) was added to the HLA-A0201 transgenic mouse-derivedsplenocytes at a final concentration of 10 μg/mL. The splenocytes addedwith the peptide were cultured for 17 hr at 37° C., 5% CO₂, wherebypeptide re-stimulation in vitro was performed. After culture, thesupernatant was removed, and the ELISPOT plate was allowed to developcolor according to the attached protocol. The number of spots thatdeveloped color was measured by ImmunoSpot Analyzer (manufactured byC.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIGS. 11 and 12. In FIGS. 11 and 12, the vertical axisshows the number of cells that reacted in the plated cells, and thehorizontal axis shows the compound or peptide administered to the mouse.In FIG. 11, the black bar shows the results of culture of HLA-A0201transgenic mouse-derived splenocytes while being pulsed with the peptideshown by SEQ ID NO: 5, and the white bar shows the results of culturewithout pulsing. That is, the difference in the values of the black andthe white bar shows the number of peptide-specific CTL, and that theadministration of the peptide shown by SEQ ID NO: 5 or a compoundrepresented by the formula (9) resulted in the induction of CTL specificto the peptide shown by SEQ ID NO: 5 in vivo in the mouse. In FIG. 11,the value of the white bar is not detected. This means that thesplenocytes of HLA-A0201 transgenic mice did not react in the absence ofpulsing with the object peptide. As a result of this test, IFNγproduction specific to the peptide shown by SEQ ID NO: 5 was detected inthe HLA-A0201 transgenic mouse-derived splenocytes. In FIG. 11, thenumber of IFNγ-producing cells specific to the peptide shown by SEQ IDNO: 5, which were induced by the administration of a compoundrepresented by the formula (9), was higher than that of thepeptide-specific IFNγ-producing cells induced by the administration ofthe peptide shown by SEQ ID NO: 5.

In FIG. 12, furthermore, the black bar shows the results of culture ofHLA-A0201 transgenic mouse-derived splenocytes while being pulsed withpeptide shown by SEQ ID NO: 24, and the white bar show the results ofculture without pulsing. That is, the difference in the values of theblack bar and the white bar shows the number of peptide-reactive cells,and that the administration of a compound represented by the formula (9)resulted in the induction of cells reactive with the helper peptideshown by SEQ ID NO: 24 in vivo in the mouse, and administration of thepeptide represented by SEQ ID NO: 5 did not induce cells reactive withthe peptide shown by SEQ ID NO: 24 in vivo in the mouse. In FIG. 12, thevalue of the white bar is scarcely detected. This means that thesplenocytes of HLA-A0201 transgenic mice did not react in the absence ofpulsing with the object peptide.

From the above, it was clarified that a compound represented by theformula (9) can induce CTL specific to the peptide shown by SEQ ID NO: 5and CTL reactive with the helper peptide shown by SEQ ID NO: 24. It wasstrongly suggested that the compound represented by the formula (9)undergoes cleavage of disulfide bond and appropriate trimming by ERAP-1in mouse in vivo and is in fact processed into the peptides shown by SEQID NOs: 5 and 24. It was assumed that the induction of the cell reactivewith the helper peptide shown by SEQ ID NO: 24 produced from a compoundrepresented by the formula (9) enhanced induction of CTL specific to thepeptide shown by SEQ ID NO: 5, and many IFNγ-producing cells specific tothe peptide shown by SEQ ID NO: 5 were found, as compared to theadministration of the compound shown by SEQ ID NO: 5.

That is, it was clarified that a compound represented by the formula(9), which is one embodiment of the compound of the present invention,is a conjugate wherein two different kinds of peptides form a compositevia the disulfide bond shown in the formula (1), and is a WT1 cancerantigen peptide conjugate vaccine that in fact can induce CTLs andhelper peptide reactive cells in vivo.

Comparative Example 1 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse and HLA-A2402 Transgenic Mouse

The compound represented by the formula (5) synthesized in Example 1 andthe peptide shown by SEQ ID NO: 238 and 239 synthesized in ReferenceExample 8 and 9 were evaluated for the CTL induction ability by an invivo CTL induction test using HLA-A0201 transgenic mouse and HLA-A2402transgenic mouse. The compound represented by the formula (5):

wherein the bond between C and C is a disulfide bond, is as described inExperimental Example 2. The peptide shown by SEQ ID NOs: 238 and 239 isa long chain peptide wherein RMFPNAPYL (SEQ ID NO: 2), which is anHLA-A0201-restricted WT1 peptide, and CYTWNQMNL (SEQ ID NO: 4), which isHLA-A2402-restricted WT1 peptide, are linked by an amide bond.

The HLA-A0201 transgenic mouse and HLA-A2402 transgenic mouse are asdescribed in Experimental Example 2.

Whether the administration of a compound represented by the formula (5)and the peptide shown by SEQ ID NO: 238, 239 results in the induction ofCTL specific to the object peptide (SEQ ID NO: 2, 4) was judged based onthe measurement of IFNγ production by re-stimulation, with the peptide(SEQ ID NO: 2, 4), of the splenocyte derived from the above-mentionedmouse administered with a compound represented by the formula (5) andthe peptide shown by SEQ ID NO: 238, 239.

Specifically, a compound represented by the formula (5) and the peptideshown by SEQ ID NOs: 238, 239 were each dissolved in dimethyl sulfoxide(DMSO) at 40 mg/mL, further diluted with water for injection to 5 mg/mL,and emulsified by mixing with an equal amount of incomplete Freund'sadjuvant (IFA). The emulsified compound was intradermally administeredto 4 sites at the base of tail of a mouse at 250 μg/site. One weeklater, the mouse was euthanized with CO₂ gas, the spleen was isolated,and splenocytes were prepared. IFNγ ELISPOT assay kit was used for themeasurement of IFNγ production. On the previous day of splenocytepreparation, an ELISPOT plate was treated with an anti-mouse IFNγantibody, and blocked with RPMI1640 medium containing 10% FBS the nextday. The prepared HLA-A0201 transgenic mouse-derived splenocytes wereplated at 0.25×10⁶ cells/well, and HLA-A2402 transgenic mouse-derivedsplenocytes were plated at 1×10⁶ cells/well, on the blocked ELISPOTplate. Peptide (SEQ ID NO: 2, 4) was dissolved in DMSO at 40 mg/mL, andfurther diluted with RPMI1640 medium containing 10% FBS to 40 μg/mL. Thediluted peptide (SEQ ID NO: 2) was added to the HLA-A0201 transgenicmouse-derived splenocytes at a final concentration of 10 μg/mL. Inaddition, the diluted peptide (SEQ ID NO: 4) was added to the HLA-A2402transgenic mouse-derived splenocytes at a final concentration of 10μg/mL. The splenocytes added with the peptide were cultivated for 18 hrat 37° C., 5% CO₂, whereby peptide re-stimulation in vitro wasperformed. After culture, the supernatant was removed, and the ELISPOTplate was allowed to develop color according to the attached protocol.The number of spots that developed color was measured by ImmunoSpotAnalyzer (manufactured by C.T.L.).

The results of IFNγ ELISPOT assay using HLA-A0201 transgenic mouse areshown in FIG. 13, and the results of IFNγ ELISPOT assay using HLA-A2402transgenic mouse are shown in FIG. 14.

In each Figure, the vertical axis shows the number of cells that reactedin the plated cells. In FIG. 13, the black bar and the white bar showthe results of culture of HLA-A0201 transgenic mouse-derived splenocytesin the presence or absence of the object peptide represented by SEQ IDNO: 2, and in FIG. 14, the black bar and the white bar show the resultsof culture of HLA-A2402 transgenic mouse-derived splenocytes in thepresence or absence of the object peptide represented by SEQ ID NO: 4.That is, the difference in the values of the black bar and the white barshow the number of the object, each peptide-specific CTL induced in themouse in vivo by the administration of a compound represented by theformula (5) and the peptide shown by SEQ ID NO: 238, 239.

In each Figure, the value of the white bar is not detected. This meansthat the splenocytes of respective transgenic mice did not react at allin the absence of the object peptide. As a result of this test, IFNγproduction specific to the object peptide shown by SEQ ID NO: 2 wasdetected in the splenocytes derived from HLA-A0201 transgenic mouseadministered with a compound represented by the formula (5), and IFNγproduction specific to the object peptide shown by SEQ ID NO: 4 wasdetected in the splenocytes derived from HLA-A02402 transgenic mouseadministered with a compound represented by the formula (5). On theother hand, while IFNγ production specific to the object peptide shownby SEQ ID NO: 2 was detected in the splenocytes derived from HLA-A0201transgenic mouse administered with the peptide shown by SEQ ID NO: 238;however, when compared to the splenocytes derived from HLA-A2402transgenic mouse administered with a compound represented by the formula(5), the number thereof was very small. IFNγ production specific to theobject peptide shown by SEQ ID NO: 4 was detected in the splenocytesderived from HLA-A2402 transgenic mouse administered with the peptideshown by SEQ ID NO: 238. While IFNγ production specific to the objectpeptide shown by SEQ ID NO: 2 was detected in the splenocytes derivedfrom HLA-A0201 transgenic mouse administered with the peptide shown bySEQ ID NO: 239; however, when compared to the splenocytes derived fromHLA-A0201 transgenic mouse administered with a compound represented bythe formula (5), the number thereof was small. IFNγ production specificto the object peptide shown by SEQ ID NO: 4 was detected in thesplenocytes derived from HLA-A2402 transgenic mouse administered withpeptide SEQ ID NO: 239.

Therefrom, the compound represented by the formula (5) of the presentinvention has been clarified to be able to efficiently induce CTLspecific to the peptide shown by SEQ ID NO: 2 and CTL specific to thepeptide shown by SEQ ID NO: 4. On the other hand, the long chain peptideshown by SEQ ID NOs: 238, 239 could not efficiently induce both the CTLspecific to the peptide shown by SEQ ID NO: 2 and the CTL specific tothe peptide shown by SEQ ID NO: 4.

Comparative Example 2

Evaluation of in vivo CTL induction ability using HLA-A0201 transgenicmouse and HLA-A2402 transgenic mouse

The compound represented by the formula (5) synthesized in Example 1 andpeptides shown by SEQ ID NOs: 240 and 241 synthesized in ReferenceExamples 10 and 11 were evaluated for the CTL induction ability by an invivo CTL induction test using HLA-A0201 transgenic mouse and HLA-A2402transgenic mouse. The compound represented by the formula (5):

wherein the bond between C and C is a disulfide bond, is as described inExperimental Example 2. The peptide shown by SEQ ID NOs: 240 and 241 isa long chain peptide wherein RMFPNAPYL (SEQ ID NO: 2), which is anHLA-A0201-restricted WT1 peptide, and CYTWNQMNL (SEQ ID NO: 4), which isan HLA-A2402-restricted WT1 peptide, are linked by an amide bond via 6glycines as a peptide spacer.

HLA-A0201 transgenic mouse and HLA-A2402 transgenic mouse are asindicated in Experimental Example 2.

Whether the administration of a compound represented by the formula (5)and SEQ ID NO: 240, 241 peptide shown by results in the induction of CTLspecific to the object peptide (SEQ ID NO: 2, 4) was judged based on themeasurement of IFNγ production by re-stimulation, with the peptide (SEQID NO: 2, 4), of the splenocyte derived from the above-mentioned mouseadministered with a compound represented by the formula (5) and thepeptide shown by SEQ ID NO: 240, 241.

Specifically, a compound represented by the formula (5) was dissolved indimethyl sulfoxide (DMSO) at 80 mg/mL, further diluted with water forinjection to 10 mg/mL, and emulsified by mixing with an equal amount ofincomplete Freund's adjuvant (IFA). The emulsified compound wasintradermally administered to 2 sites at the base of tail of a mouse at500 μg/site. In addition, the peptides shown by SEQ ID NOs: 240, 241were dissolved in dimethyl sulfoxide (DMSO) at 80 mg/mL, further dilutedwith water for injection to 11 mg/mL, and emulsified by mixing with anequal amount of incomplete Freund's adjuvant (IFA). The emulsifiedcompound was intradermally administered to 2 sites at the base of tailof a mouse at 550 μg/site. One week later, the mouse was euthanized withCO₂ gas, the spleen was isolated, and splenocytes were prepared. IFNγELISPOT assay kit was used for the measurement of IFNγ production. Onthe previous day of splenocyte preparation, an ELISPOT plate was treatedwith an anti-mouse IFNγ antibody, and blocked with RPMI1640 mediumcontaining 10% FBS the next day. The prepared HLA-A0201 transgenicmouse-derived splenocytes at 0.25×10⁶ cells/well and HLA-A2402transgenic mouse-derived splenocytes at 1.5×10⁶ cells/well were platedon the blocked ELISPOT plate. Peptide (SEQ ID NO: 2, 4) was dissolved inDMSO at 40 mg/mL, and further diluted with RPMI1640 medium containing10% FBS to 40 μg/mL. The diluted peptide (SEQ ID NO: 2) was added to theHLA-A0201 transgenic mouse-derived splenocytes at a final concentrationof 10 μg/mL. In addition, the diluted peptide (SEQ ID NO: 4) was addedto HLA-A2402 transgenic mouse-derived splenocytes at a finalconcentration of 10 μg/mL. The splenocytes added with the peptide werecultured for 17 hr at 37° C., 5% CO₂, whereby peptide re-stimulation invitro was performed. After culture, the supernatant was removed, and theELISPOT plate was allowed to develop color according to the attachedprotocol. The number of spots that developed color was measured byImmunoSpot Analyzer (manufactured by C.T.L.).

The results of IFNγ ELISPOT assay using HLA-A0201 transgenic mouse areshown in FIG. 15, and the results of IFNγ ELISPOT assay using HLA-A2402transgenic mouse are shown in FIG. 16.

In each Figure, the vertical axis shows the number of cells that reactedin the plated cells. In FIG. 15, the black bar and the white bar showthe results of culture of HLA-A0201 transgenic mouse-derived splenocytesin the presence or absence of the object peptide represented by SEQ IDNO: 2, and in FIG. 16, the black bar and the white bar show the resultsof culture of HLA-A2402 transgenic mouse-derived splenocytes in thepresence or absence of the object peptide represented by SEQ ID NO: 4.That is, the difference in the values of the black bar and the white barshow the number of the object, each peptide-specific CTL induced in themouse in vivo by the administration of a compound represented by theformula (5) and the peptides shown by SEQ ID NOs: 240, 241.

In each Figure, the value of the white bar is not detected. This meansthat the splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered with acompound represented by the formula (5), and IFNγ production specific tothe object peptide shown by SEQ ID NO: 4 was detected in the splenocytesderived from HLA-A2402 transgenic mouse administered with a compoundrepresented by the formula (5). On the other hand, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was extremely lessin the splenocytes derived from HLA-A0201 transgenic mouse administeredwith the peptide shown by SEQ ID NO: 240; however, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 4 was detected in thesplenocytes derived from HLA-A02402 transgenic mouse administered with acompound represented by SEQ ID NO: 240. In addition, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was extremely lessin the splenocytes derived from HLA-A0201 transgenic mouse administeredwith the peptide shown by SEQ ID NO: 241. While IFNγ production specificto the object peptide shown by SEQ ID NO: 4 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered withthe peptide shown by SEQ ID NO: 241; however, when compared to thesplenocytes derived from HLA-A2402 transgenic mouse administered with acompound represented by the formula (5), the number thereof was verysmall.

Therefrom, the compound represented by the formula (5) of the presentinvention has been clarified to be able to efficiently induce CTLspecific to the peptide shown by SEQ ID NO: 2 and CTL specific to thepeptide shown by SEQ ID NO: 4. On the other hand, the long chain peptidecontaining the peptide spacer shown by SEQ ID NO: 240, 241 could notefficiently induce both the CTL specific to the peptide shown by SEQ IDNO: 2 and the CTL specific to the peptide shown by SEQ ID NO: 4.

Experimental Example 10

The peptide synthesized in Reference Example 3 and the compounds(conjugates) synthesized in Examples 6 and 9 were subjected to thesolubility measurement by a method similar to that in ExperimentalExample 3. Each solubility is shown in Table 66.

TABLE 66 Ex. No. SEQ ID NO: and amino acid sequence or or formula pH 6.0pH 7.4 Ref. Ex. structural formula No. (mg/mL) (mg/mL) Ref.CNKRYFKLSHLQMHSRKH SEQ ID NO: 23 >1.0 0.712 Ex. 3 Ex.6

formula (3) >1.0 >1.0 Ex. 10

formula (9) >1.0 0.565

Examples 11-12

By a method similar to that in Example 2, peptides consisting of theamino acid sequences of SEQ ID NOs: 242-243 were synthesized. Theresults of mass spectrometry are shown in Table 67. The peptidesdescribed in Table 67 are the compounds of the present invention.

TABLE 67 mass mass spectro- spectro- SEQ metry: metry: Ex. amino acid IDLC-ESI/MS Calcu- No. sequence NO: m/z lated 11 CWAPVLDFAPPGASAYGSL 2421923.5 1923.2 [M + H]¹⁺ 12 WAPVLDFAPPGASAYGSLC 243 1923.6 1923.2 [M +H]¹⁺

Example 13

By a method similar to that in Example 1, each compound (conjugate)represented by the formula 10 was synthesized. The results of massspectrometry are shown in Table 68, wherein the bond between C and C isa disulfide bond.

TABLE 68 mass mass spec- spectrom- trom- form- etry: etry: Ex. ulaLC-ESI/ Calcu- No. structural formula No. MS m/z lated 13

10 1566.6 [M + 2H]²⁺ 1566.8

Reference Example 12

By a method similar to that in Example 1, each compound (conjugate)represented by the formula 11 was synthesized. The results of massspectrometry are shown in Table 69, wherein the bond between C and C isa disulfide bond. The peptide described in the Table is not the compoundof the present invention, and therefore, it is described as ReferenceExample.

TABLE 69 mass spectrom- mass Ref. etry: spectrom- Ex. formula LC-ESI/MSetry: No. structural formula No. m/z Calculated 12

11 1044.8 [M + 3H]³⁺ 1044.9

Example 14 Synthesis of the Compound Represented by the Formula (12)

wherein the bond between C and C is a disulfide bond

Step 1. Synthesis of Fmoc-Cys(Mmt)-Ala-SBn (Mmt is 4-Methoxytrityl)

(Synthesis of Fmoc-C(Mmt)A-SBn)

A solution of Fmoc-Cys(Mmt)-OH (4.80 g), N,N-diisopropylethylamine (2.56mL), hexafluorophosphoric acid(benzotriazol-1-yloxy)tripyrrolidinophosphonium (4.50 g) and H-Ala-SBnsynthesized by a known method (for example, Journal of OrganicChemistry, Vol. 64, No. 24 8761-8769) in chloroform (20 ml) was stirredat room temperature for 1 hr. The reaction mixture was purified bycolumn chromatography (elution solvent, hexane/ethyl acetate) to givethe object compound, Fmoc-C(Mmt)A-SBn (2.80 g).

NMR: ¹H NMR (CDCl₃) δ 7.72 (t, J=7.6 Hz, 2H), 7.54 (d, J=7.2 Hz, 1H),7.38-7.34 (m, 7H), 7.29-7.25 (m, 6H), 7.23-7.15 (m, 7H), 6.76 (d, J=8.8Hz, 2H), 6.15 (d, J=8.0 Hz, 1H), 4.95 (d, J=7.2 Hz, 1H), 4.57 (quin,J=7.6 Hz, 1H), 4.35 (d, J=6.8 Hz, 2H) 4.19-4.17 (m, 1H), 4.04 (s, 2H),3.73 (s, 3H), 2.72 (dd, J=13.2, 8.4 Hz, 1H), 2.61 (d, J=9.6 Hz, 1H),1.31 (d, J=7.2 Hz, 3H).

Step 2. Synthesis ofH-Cys(Mmt)-Ala-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH (Synthesis ofC(Mmt)ACYTWNQMNL)

A solution of Fmoc-Cys(Mmt)-Ala-SBn (11 mg) obtained in step 1,H-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH (21 mg) synthesized by a knownmethod (for example, WO07/063903), N,N-diisopropylethylamine (200 μL),3,3′,3″-Phosphanetriyl tripropanoic acid hydrochloride (1 mg),4-mercaptophenylacetic acid (1 mg) and 0.1M sodium phosphate buffer (pH7.5, 200 μL) in DMF (400 μL) was stirred at room temperature for 4 hr.To the reaction mixture was added diethylamine (200 μL) and the mixturewas further stirred for 15 min. The reaction mixture was purified byreversed-phase HPLC to give the object compound, C(Mmt)ACYTWNQMNL (7mg).

mass spectrometry: LC-ESI/MS m/z=810.2 [M+2H]²⁺ (Calculated=810.5)

Step 3. Synthesis of(H-Cys(Mmt)-Ala-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH)(H-Cys-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH) Disulfide Bond

[i.e., synthesis of a compound represented by the formula (13):

wherein the bond between C and C is a disulfide bond.

A solution of H-Cys(Mmt)-Ala-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH (51mg) obtained in step 2 and(H-Cys(Npys)-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH (43 mg) obtained inExample 1, step 1 in DMF (4 mL) was stirred at room temperature for 2hr. The reaction mixture was purified by reversed-phase HPLC to give 39mg of the object compound,(H-Cys(Mmt)-Ala-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH)(H-Cys-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH) disulfide bond [i.e., acompound represented by the formula (13)].

mass spectrometry: LC-ESI/MS m/z=1414.4 [M+2H]²⁺ (Calculated=1415.2)

Step 4. Synthesis ofH-Cys(SPy)-Asn-Lys-Arg-Tyr-Phe-Lys-Leu-Ser-His-Leu-Gln-Met-His-Ser-Arg-Lys-OH

(Synthesis of C(SPy)NKRYFKLSHLQMHSRK)

A 20% w/w solution ofH-Cys-Asn-Lys-Arg-Tyr-Phe-Lys-Leu-Ser-His-Leu-Gln-Met-His-Ser-Arg-Lys-OH(182 mg) obtained in Reference Example 1 and 2,2′-dipyridylbisulfide(0.2M isopropanol solution, 544 μL) in acetic acid water (4 mL) wasstirred at room temperature for 17 hr. The reaction mixture was purifiedby reversed-phase HPLC to give the object compound,H-Cys(SPy)-Asn-Lys-Arg-Tyr-Phe-Lys-Leu-Ser-His-Leu-Gln-Met-His-Ser-Arg-Lys-OH(177 mg).

mass spectrometry: LC-ESI/MS m/z=1143.5 [M+21-1]²⁺ (Calculated=1142.9)

Step 5. Synthesis of a Compound Represented by the Formula (12)

wherein the bond between C and C is a disulfide bond

A solution of(H-Cys(Mmt)-Ala-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH)(H-Cys-Arg-Met-Phe-Pro-Asn-Ala-Pro-Tyr-Leu-OH)disulfide bond obtained in step 3 [i.e., a compound represented by theformula (13)] (9 mg),H-Cys(SPy)-Asn-Lys-Arg-Tyr-Phe-Lys-Leu-Ser-His-Leu-Gln-Met-His-Ser-Arg-Lys-OH(24 mg) obtained in step 4 and triisopropylsilane (10 μL) intrifluoroacetic acid (190 μL) was stirred at room temperature for 1 hr.The reaction mixture was purified by reversed-phase HPLC to give theobject compound, a compound represented by the formula 12 (5 mg).

mass spectrometry: LC-ESI/MS m/z=1577.2 [M+3H]³⁺ (Calculated=1577.9)

Examples 15-16

By a method similar to that in Example 14, each compound (conjugate)represented by the formula 14 or 15 was synthesized. The results of massspectrometry are shown in Table 70, wherein the bond between C and C isa disulfide bond.

TABLE 70 mass spectrom- etry: mass Ex. formula LC-ESI/MS spectrometry:No. structural formula No. m/Z Caluclated 15

14 1492.5 [M + 3H]³⁺ 1493.1 16

15 1492.5 [M + 3H]³⁺ 1493.1

Reference Example 13

By a method similar to that in Example 2, peptides consisting of theamino acid sequence of SEQ ID NO: 244 were synthesized. Table 71 showsthe results of mass spectrometry. The peptide described in the Table isnot the compound of the present invention, and therefore, it isdescribed as Reference Example.

TABLE 71 mass spectro- mass Ref. SEQ metry: spectro- Ex. amino acid IDLC-ESI/MS metry: No. sequence NO: m/Z Calculated 13 WAPVLDFAPPGASAYGSL244 1819.8 1819.1 [M + H]⁺

Experimental Example 11 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse

The compound represented by the formula (10) synthesized in Example 13was evaluated for the CTL induction ability by an in vivo CTL inductiontest using HLA-A0201 transgenic mouse. The compound represented by theformula (10):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is RMFPNAPYL (SEQ ID NO: 2) and cancer antigen peptide B isWAPVLDFAPPGASAYGSL (SEQ ID NO: 244). RMFPNAPYL (SEQ ID NO: 2) is anHLA-A0201-restricted WT1 peptide, and WAPVLDFAPPGASAYGSL (SEQ ID NO:244) is MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (10)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (10). Whether or not the helper peptide (SEQID NO: 244) works in the living body was judged by comparison of thenumber of IFNγ-producing cells when the splenocytes derived from theabove-mentioned mouse administered with a compound represented by theformula (10) and the splenocytes derived from the above-mentioned mouseadministered with the peptide shown by SEQ ID NO: 2 were re-stimulatedwith the peptide (SEQ ID NO: 2).

Specifically, a compound shown by SEQ ID NO: 2 was dissolved in dimethylsulfoxide (DMSO) at 80 mg/mL, further diluted with water for injectionto 3 mg/mL, and emulsified by mixing with an equal amount of incompleteFreund's adjuvant (IFA). The emulsified compound was intradermallyadministered to 2 sites at the base of tail of a mouse at 150 μg/site.In addition, a compound represented by the formula (10) was dissolved indimethyl sulfoxide (DMSO) at 80 mg/mL, further diluted with water forinjection to 8.5 mg/mL, and emulsified by mixing with an equal amount ofincomplete Freund's adjuvant (IFA). The emulsified compound wasintradermally administered to 2 sites at the base of tail of a mouse at425 μg/site. The mole number of the peptide of the SEQ ID NO: 2contained in the dose of a compound represented by the formula (10) permouse was controlled to be equal to the mole number contained in thedose of the peptide shown by SEQ ID NO: 2 per mouse. In addition, theconcentration of DMSO contained in each emulsion was also set to thesame level. One week later, the mouse was euthanized with CO₂ gas, thespleen was isolated, and splenocytes were prepared. IFNγ ELISPOT assaykit was used for the measurement of IFNγ production. On the previous dayof splenocyte preparation, an ELISPOT plate was treated with ananti-mouse IFNγ antibody, and blocked with RPMI1640 medium containing10% FBS the next day. The prepared HLA-A0201 transgenic mouse-derivedsplenocytes were plated at 0.125×10⁶ cells/well on the blocked ELISPOTplate. Peptide (SEQ ID NO: 2, 4) was dissolved in DMSO at 40 mg/mL, andfurther diluted with RPMI1640 medium containing 10% FBS to 40 μg/mL. Thediluted peptide (SEQ ID NO: 2) was added to the HLA-A0201 transgenicmouse-derived splenocytes at a final concentration of 10 μg/mL. Thesplenocytes added with the peptide were cultivated for 19 hr at 37° C.,5% CO₂, whereby peptide re-stimulation in vitro was performed. Afterculture, the supernatant was removed, and the ELISPOT plate was allowedto develop color according to the attached protocol. The number of spotsthat developed color was measured by ImmunoSpot Analyzer (manufacturedby C.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 17. In FIG. 17, the vertical axis shows the number ofcells that reacted in the plated cells, and the horizontal axis showscompound or peptide administered to the mouse. In FIG. 17, the black barshows the results of culture of HLA-A0201 transgenic mouse-derivedsplenocytes while being pulsed with the peptide shown by SEQ ID NO: 2,and the white bar shows the results of culture without pulsing. That is,the difference in the values of the black bar and the white bar showsthe number of peptide-specific CTL, and that the administration of thepeptide shown by SEQ ID NO: 2 or a compound represented by the formula(10) resulted in the induction of CTL specific to the peptide shown bySEQ ID NO: 2 in vivo in the mouse. In FIG. 17, the value of the whitebar is not detected. This means that the splenocytes of HLA-A0201transgenic mice did not react at all in the absence of pulsing with theobject peptide. As a result of this test, IFNγ production specific tothe peptide shown by SEQ ID NO: 2 was is detected in the HLA-A0201transgenic mouse-derived splenocytes. Moreover, in FIG. 17, the numberof IFNγ-producing cells specific to the peptide shown by SEQ ID NO: 2,which were induced by the administration of a compound represented bythe formula (10), was higher than that of the peptide-specificIFNγ-producing cells induced by the administration of the peptide shownby SEQ ID NO: 2.

From the above, it was clarified that a compound represented by theformula (10) can induce CTL specific to the peptide shown by SEQ ID NO:2. When a compound represented by the formula (10) was administered,many IFNγ producing cells specific to the peptide shown by SEQ ID NO: 2were observed as compared to administration of the peptide shown by SEQID NO: 2. It was assumed that the induction of the cell reactive withthe helper peptide shown by SEQ ID NO: 244 produced from a compoundrepresented by the formula (10) enhanced induction of CTL specific tothe peptide shown by SEQ ID NO: 2. Therefore, it was strongly suggestedthat the compound represented by the formula (10) undergoes cleavage ofdisulfide bond and appropriate trimming by ERAP-1 in mouse in vivo andis in fact processed into the peptides shown by SEQ ID NOs: 2 and 244.

That is, it was clarified that a compound represented by the formula(10), which is one embodiment of the compound of the present invention,is a conjugate wherein two different kinds of peptides form a compositevia the disulfide bond shown in the formula (1), and is a WT1 cancerantigen peptide conjugate vaccine that in fact can induce CTLs andhelper peptide reactive cells in vivo.

Comparative Example 3 Evaluation of In Vivo CTL Induction Ability UsingHLA-A0201 Transgenic Mouse

The compound represented by the formula (11) synthesized in ReferenceExample 12 was evaluated for the CTL induction ability by an in vivo CTLinduction test using HLA-A0201 transgenic mouse. The compoundrepresented by the formula (11):

wherein the bond between C and C is a disulfide bond, is, in particular,a compound of the aforementioned formula (1), wherein cancer antigenpeptide A is RMFPNAPYL (SEQ ID NO: 2) and cancer antigen peptide C isWAPVLDFAPPGASAYGSLC (SEQ ID NO: 243). RMFPNAPYL (SEQ ID NO: 2) is aHLA-A0201-restricted WT1 peptide, and WAPVLDFAPPGASAYGSL (SEQ ID NO:244) is an MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (11)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2), of the splenocytesderived from the above-mentioned mouse administered with a compoundrepresented by the formula (11). Whether or not the helper peptide (SEQID NO: 244) works in the living body was judged by comparison of thenumber of IFNγ-producing cells when the splenocytes derived from theabove-mentioned mouse administered with a compound represented by theformula (11) and the splenocytes derived from the above-mentioned mouseadministered with the peptide shown by SEQ ID NO: 2 were re-stimulatedwith the peptide (SEQ ID NO: 2).

By a method similar to that in Experimental Example 11, CTL inductiontest was performed.

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 18. In FIG. 18, the vertical axis shows the number ofcells that reacted in the plated cells, and the horizontal axis showscompound or peptide administered to the mouse. In FIG. 18, the black barshows the results of culture of HLA-A0201 transgenic mouse-derivedsplenocytes while being pulsed with the peptide shown by SEQ ID NO: 2,and the white bar shows the results of culture without pulsing. That is,the difference in the values of the black bar and the white bar showsthe number of peptide-specific CTL, and that the administration of thepeptide shown by SEQ ID NO: 2 or a compound represented by the formula(11) resulted in the induction of CTL specific to the peptide shown bySEQ ID NO: 2 in vivo in the mouse. In FIG. 18, the value of the whitebar is not detected. This means that the splenocytes of HLA-A0201transgenic mice did not react at all in the absence of pulsing with theobject peptide. As a result of this test, IFNγ production specific tothe peptide shown by SEQ ID NO: 2 was detected in the HLA-A0201transgenic mouse-derived splenocytes. On the other hand, in FIG. 18, anincrease in the IFNγ producing cells specific to the peptide shown bySEQ ID NO: 2, which was induced by the administration of the peptideshown by SEQ ID NO: 2, could not be detected by the administration of acompound represented by the formula (11).

The results of Experimental Example 11 and Comparative Example 3 suggestthat, when WAPVLDFAPPGASAYGSL (SEQ ID NO: 244) is used as an MHC classII-restricted WT1 peptide, WAPVLDFAPPGASAYGSL (SEQ ID NO: 244) as thecancer antigen peptide B in the aforementioned formula (1) is a morepreferable embodiment of the invention than WAPVLDFAPPGASAYGSLC (SEQ IDNO: 243) as the cancer antigen peptide C.

Experimental Example 12 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse and HLA-A2402 Transgenic Mouse

The CTL induction ability of the compound represented by the formula 12synthesized in Example 14 was evaluated by an in vivo CTL induction testusing an HLA-A0201 transgenic mouse and an HLA-A2402 transgenic mouse.RMFPNAPYL (SEQ ID NO: 2) contained in the compound represented by theformula (12):

wherein the bond between C and C is a disulfide bond, is anHLA-A0201-restricted WT1 peptide, CYTWNQMNL (SEQ ID NO: 4) isHLA-A2402-restricted WT1 peptide, and CNKRYFKLSHLQMHSRK (SEQ ID NO: 22)is MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse and HLA-A2402 transgenic mouse are asdescribed in Experimental Examples 2 and 5.

Whether the administration of a compound represented by the formula (12)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2, 4) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2, 4), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (12). Whether or not the helper peptide (SEQID NO: 22) works in the living body was judged by comparison of thenumber of IFNγ-producing cells when the splenocytes derived from theabove-mentioned mouse administered with a compound represented by theformula (12) and the splenocytes derived from the above-mentioned mouseadministered with a compound represented by the formula (5) werere-stimulated with the peptide (SEQ ID NOs: 2, 4).

Specifically, a compound represented by the formula (5) was dissolved indimethyl sulfoxide (DMSO) at 80 mg/mL, further diluted with water forinjection to 3 mg/mL, and emulsified by mixing with an equal amount ofMontanide ISA51VG. The emulsified compound was intradermallyadministered to 2 sites at the base of tail of a mouse at 150 μg/site.In addition, a compound represented by the formula (12) was dissolved indimethyl sulfoxide (DMSO) at 80 mg/mL, further diluted with water forinjection to 6 mg/mL, and emulsified by mixing with an equal amount ofMontanide ISA51VG. The emulsified compound was intradermallyadministered to 2 sites at the base of tail of a mouse at 300 μg/site.The mole number of the compound represented by the formula (5) containedin the dose of the compound represented by the formula (12) per mousewas controlled to be equal to the mole number contained in the dose ofthe compound represented by the formula (5) per mouse. In addition, theconcentration of DMSO contained in each emulsion was also set to thesame level. One week later, the mouse was euthanized with CO₂ gas, thespleen was isolated, and splenocytes were prepared. IFNγ ELISPOT assaykit was used for the measurement of IFNγ production. On the previous dayof splenocyte preparation, an ELISPOT plate was treated with ananti-mouse IFNγ antibody, and blocked with RPMI1640 medium containing10% FBS the next day. The prepared HLA-A0201 transgenic mouse-derivedsplenocytes were plated, and HLA-A2402 transgenic mouse-derivedsplenocytes were each plated at 0.25×10⁶ cells/well, on the blockedELISPOT plate. Peptide (SEQ ID NO: 2, 4) was dissolved in DMSO at 40mg/mL, and further diluted with RPMI1640 medium containing 10% FBS to 40μg/mL. The diluted peptide (SEQ ID NO: 2) was added to the HLA-A0201transgenic mouse-derived splenocytes at a final concentration of 10μg/mL. In addition, the diluted peptide (SEQ ID NO: 4) was added to theHLA-A2402 transgenic mouse-derived splenocytes at a final concentrationof 10 μg/mL. The splenocytes added with the peptide were cultivated for17 hr at 37° C., 5% CO₂, whereby peptide re-stimulation in vitro wasperformed. After culture, the supernatant was removed, and the ELISPOTplate was allowed to develop color according to the attached protocol.The number of spots that developed color was measured by ImmunoSpotAnalyzer (manufactured by C.T.L.).

The results of IFNγ ELISPOT assay using HLA-A0201 transgenic mouse areshown in FIG. 19, and the results of IFNγ ELISPOT assay using HLA-A2402transgenic mouse are shown in FIG. 20.

In each Figure, the vertical axis shows the number of cells that reactedin the plated cells. In FIG. 19, the black bar and the white bar showthe results of culture of HLA-A0201 transgenic mouse-derived splenocytesin the presence or absence of the object peptide represented by SEQ IDNO: 2, and in FIG. 20, the black bar and the white bar show the resultsof culture of HLA-A2402 transgenic mouse-derived splenocytes in thepresence or absence of the object peptide represented by SEQ ID NO: 4.That is, the difference in the values of the black bar and the white barshow the number of the object, each peptide-specific CTL induced in themouse in vivo by the administration of compounds represented by theformula (5) and formula (12).

In each Figure, the value of the white bar is not detected. This meansthat the splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in theHLA-A0201 transgenic mouse-derived splenocytes administered compoundsrepresented by the formula (5) and formula (12), and IFNγ productionspecific to the object peptide shown by SEQ ID NO: 4 was detected in theHLA-A2402 transgenic mouse-derived splenocytes administered compoundsrepresented by the formula (5) and formula (12). In FIG. 19, the numberof the IFNγ-producing cells specific to the peptide shown by SEQ ID NO:2, which was induced by the administration of a compound represented bythe formula (12), was higher than the number of the IFNγ producing cellsspecific to peptide, which was induced by the administration of acompound represented by the formula (5). On the other hand, in FIG. 20,the number of the IFNγ-producing cells specific to the peptide shown bySEQ ID NO: 4, which was induced by the administration of a compoundrepresented by the formula (12), did not differ much from the number ofthe IFNγ producing cells specific to peptide, which was induced by theadministration of a compound represented by the formula (5).

From the above, it was clarified that a compound represented by theformula (12) can induce CTL specific to the peptide shown by SEQ ID NOs:2, 4. When a compound represented by the formula (12) was administered,many IFNγ producing cells specific to the peptide shown by SEQ ID NO: 2were observed as compared to administration of the compound representedby the formula (5). It was assumed that the induction of the cellreactive with the helper peptide shown by SEQ ID NO: 22 produced from acompound represented by the formula (12) enhanced induction of CTLspecific to the peptide shown by SEQ ID NO: 2. The absence of muchdifference in the number of IFNγ-producing cells specific to the peptideshown by SEQ ID NO: 4 between the administration of a compoundrepresented by the formula 12 and the administration of a compoundrepresented by the formula (5) was assumed to be attributable to theabsence of induction of the cells reactive with the helper peptide shownby SEQ ID NO: 22, since the HLA-A2402 transgenic mouse does not expresshuman MHC class II. Accordingly, it was strongly suggested that thecompound represented by the formula (12) undergoes cleavage of disulfidebond and appropriate trimming by ERAP-1 in mouse in vivo and is in factprocessed into the peptides shown by SEQ ID NOs: 2, 4 and 22.

That is, it was clarified that a compound represented by the formula(12), which is one embodiment of the compound of the present invention,is a conjugate wherein three different kinds of peptides form acomposite via the disulfide bond, and is a WT1 cancer antigen peptideconjugate vaccine that in fact can induce CTLs and helper peptidereactive cells in vivo.

Experimental Example 13 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse and HLA-A2402 Transgenic Mouse

The CTL induction ability of the compound represented by the formula(14) synthesized in Example 15 was evaluated by an in vivo CTL inductiontest using an HLA-A0201 transgenic mouse and an HLA-A2402 transgenicmouse. RMFPNAPYL (SEQ ID NO: 2) contained in a compound represented bythe formula (14):

wherein the bond between C and C is a disulfide bond, is anHLA-A0201-restricted WT1 peptide, CYTWNQMNL (SEQ ID NO: 4) isHLA-A2402-restricted WT1 peptide, WAPVLDFAPPGASAYGSL (SEQ ID NO: 244) isMHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse and HLA-A2402 transgenic mouse are asdescribed in Experimental Examples 2 and 5.

Whether the administration of a compound represented by the formula (14)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2, 4) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2, 4), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (14). Whether or not helper peptide (SEQ IDNO: 244) works in the living body was judged by comparison of the numberof IFNγ-producing cells when the splenocytes derived from theabove-mentioned mouse administered with a compound represented by theformula (14) and the splenocytes derived from the above-mentioned mouseadministered with a compound represented by the formula (5) werere-stimulated with the peptide (SEQ ID NO: 2).

By a method similar to that in Experimental Example 12, a CTL inductiontest was performed. The compound represented by the formula (14) wasdissolved in dimethyl sulfoxide (DMSO) at 80 mg/mL, diluted with waterfor injection at 5.6 mg/mL, and mixed with an equal amount of MontanideISA51VG to give an emulsion. The emulsified compound was intradermallyadministered to 2 sites at the base of tail of a mouse at 280 μg/site.

The results of IFNγ ELISPOT assay using HLA-A0201 transgenic mouse areshown in FIG. 21, and the results of IFNγ ELISPOT assay using HLA-A2402transgenic mouse are shown in FIG. 22.

In each Figure, the vertical axis shows the number of cells that reactedin the plated cells. In FIG. 21, the black bar and the white bar showthe results of culture of HLA-A0201 transgenic mouse-derived splenocytesin the presence or absence of the object peptide represented by SEQ IDNO: 2, and in FIG. 22, the black bar and the white bar show the resultsof culture of HLA-A2402 transgenic mouse-derived splenocytes in thepresence or absence of the object peptide represented by SEQ ID NO: 4.That is, the difference in the values of the black bar and the white barshow the number of the object, each peptide-specific CTL induced in themouse in vivo by the administration of compounds represented by theformulas (5) and (14).

In each Figure, the value of the white bar is not detected. This meansthat the splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered withcompounds represented by the formulas (5) and (14), and IFNγ productionspecific to the object peptide shown by SEQ ID NO: 4 was detected in thesplenocytes derived from HLA-A2402 transgenic mouse administered withcompounds represented by the formulas (5) and (14). In FIGS. 21 and 22,the number of the IFNγ producing cells specific to the peptide shown bySEQ ID NO: 2, 4, which was induced by the administration of a compoundrepresented by the formula (14), was higher than the number of thepeptide-specific IFNγ producing cells, which was induced by theadministration of a compound represented by the formula (5).

From the above, it was clarified that a compound represented by theformula (14) can induce CTL specific to the peptides shown by SEQ IDNOs: 2 and 4. It was assumed that the induction of the cell reactivewith the helper peptide shown by SEQ ID NO: 244 produced from a compoundrepresented by the formula (14) enhanced induction of CTL specific tothe peptide shown by SEQ ID NO: 2, and many IFNγ-producing cellsspecific to the peptide shown by SEQ ID NO: 2 were found when a compoundrepresented by the formula (14) was administered as compared to theadministration of a compound represented by the formula (5). On theother hand, many IFNγ-producing cells specific to the peptide shown bySEQ ID NO: 4 were found when a compound represented by the formula (14)was administered as compared to the administration of a compoundrepresented by the formula (5). It was assumed that the peptide shown bySEQ ID NO: 244 was bound to mouse MHC class II expressed in HLA-A2402transgenic mouse to induce the cell reactive with the helper peptide,which in turn enhanced induction of CTL specific to the peptide shown bySEQ ID NO: 4. Therefore, it was strongly suggested that the compoundrepresented by the formula (14) undergoes cleavage of disulfide bond andappropriate trimming by ERAP-1 in mouse in vivo and is in fact processedinto the peptides shown by SEQ ID NOs: 2, 4 and 244.

That is, it was clarified that a compound represented by the formula(14), which is one embodiment of the compound of the present invention,is a conjugate wherein three different kinds of peptides form acomposite via the disulfide bond, and is a WT1 cancer antigen peptideconjugate vaccine that in fact can induce CTLs and helper peptidereactive cells in vivo.

Experimental Example 14 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse

A cocktail vaccine which is a mixture of the compound represented by theformula (5) synthesized in Example 1 and the peptide shown by SEQ ID NO:22 synthesized in Reference Example 1 was evaluated for the CTLinduction ability by an in vivo CTL induction test using HLA-A0201transgenic mouse. RMFPNAPYL (SEQ ID NO: 2) contained in the compoundrepresented by the formula (5):

wherein the bond between C and C is a disulfide bond, is anHLA-A0201-restricted WT1 peptide, CYTWNQMNL (SEQ ID NO: 4) isHLA-A2402-restricted WT1 peptide, and CNKRYFKLSHLQMHSRK (SEQ ID NO: 22)is MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (5)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (5). Whether or not the helper peptide (SEQID NO: 22) mixed with the formula (5) works in the living body wasjudged by comparison of the number of IFNγ-producing cells when thesplenocytes derived from the above-mentioned mouse administered with acompound represented by the formula (5) alone and the splenocytesderived from the above-mentioned mouse administered with a cocktailvaccine of a compound represented by the formula (5) and the peptideshown by SEQ ID NO: 22 were re-stimulated with the peptide (SEQ ID NO:2).

Specifically, a compound represented by the formula (5) was dissolved indimethyl sulfoxide (DMSO) at 80 mg/mL, further diluted with water forinjection to 3 mg/mL, and emulsified by mixing with an equal amount ofMontanide ISA51VG. The emulsified compound was intradermallyadministered to 2 sites at the base of tail of a mouse at 150 μg/site.In addition, a compound represented by the formula (5) and the peptideshown by SEQ ID NO: 22 were dissolved in dimethyl sulfoxide (DMSO) at 80mg/mL, diluted with water for injection and mixed such that theconcentration after dilution was 3 mg/mL for the compound represented bythe formula (5), and 2.7 mg/mL for the peptide shown by SEQ ID NO: 22.The diluted solution was mixed with an equal amount of Montanide ISA51VGto give an emulsion. The cocktail vaccine containing a compoundrepresented by the formula (5) at 150 μg/site, and the peptide shown bySEQ ID NO: 22 at 137 μg/site was intradermally administered to 2 sitesat the base of tail of a mouse. The DMSO concentration of each emulsionwas set to the same level. One week later, the mouse was euthanized withCO₂ gas, the spleen was isolated, and splenocytes were prepared. IFNγELISPOT assay kit was used for the measurement of IFNγ production. Onthe previous day of splenocyte preparation, an ELISPOT plate was treatedwith an anti-mouse IFNγ antibody, and blocked with RPMI1640 mediumcontaining 10% FBS the next day. The prepared HLA-A0201 transgenicmouse-derived splenocytes were plated at 0.25×10⁶ cells/well on theblocked ELISPOT plate. Peptide (SEQ ID NO: 2) was dissolved in DMSO at40 mg/mL, and further diluted with RPMI1640 medium containing 10% FBS to40 μg/mL. The diluted peptide (SEQ ID NO: 2) was added to the HLA-A0201transgenic mouse-derived splenocytes at a final concentration of 10μg/mL. The splenocytes added with the peptide were cultured for 17 hr at37° C., 5% CO₂, whereby peptide re-stimulation in vitro was performed.After culture, the supernatant was removed, and the ELISPOT plate wasallowed to develop color according to the attached protocol. The numberof spots that developed color was measured by ImmunoSpot Analyzer(manufactured by C.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 23.

In FIG. 23, the vertical axis shows the number of cells that reacted inthe plated cells. In FIG. 23, the black bar and the white bar show theresults of culture of HLA-A0201 transgenic mouse-derived splenocytes inthe presence or absence of the object peptide represented by SEQ ID NO:2. That is, the difference in the values of the black bar and the whitebar shows the number of the object, each peptide-specific CTL induced inthe mouse in vivo by the administration of a cocktail vaccine containinga compound represented by the formula (5) and a helper peptide (SEQ IDNO: 22).

In FIG. 23, the value of the white bar is not detected. This means thatthe splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered with acompound represented by the formula (5) alone, and a cocktail vaccinecontaining a helper peptide (SEQ ID NO: 22). In FIG. 23, the number ofthe IFNγ producing cells specific to the peptide shown by SEQ ID NO: 2,which was induced by the administration of a cocktail vaccine containinga helper peptide (SEQ ID NO: 22), was higher than the number of thepeptide-specific IFNγ producing cells, which was induced by theadministration of a compound represented by the formula (5) alone.

From the above, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the peptide shown by SEQ IDNO: 22 can induce CTL specific to the peptides shown by SEQ ID NO: 2. Inaddition, many IFNγ-producing cells specific to the peptide shown by SEQID NO: 2 were found when a cocktail vaccine was administered as comparedto the single administration of a compound represented by the formula(5). It was assumed that the induction of the cell reactive with thehelper peptide shown by SEQ ID NO: 22 contained in the cocktail vaccineenhanced induction of CTL specific to the peptide shown by SEQ ID NO: 2.Therefore, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the helper peptide canstrongly induce CTL in the body of mouse as compared to the singleadministration of a compound represented by the formula (5).

Experimental Example 15 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse

The CTL induction ability of a cocktail vaccine of a compoundrepresented by the formula (5) synthesized in Example 1 and the peptideshown by SEQ ID NO: 244 synthesized in Reference Example 13 wasevaluated by an in vivo CTL induction test using an HLA-A0201 transgenicmouse. RMFPNAPYL (SEQ ID NO: 2) contained in a compound represented bythe formula (5):

wherein the bond between C and C is a disulfide bond, is anHLA-A0201-restricted WT1 peptide, CYTWNQMNL (SEQ ID NO: 4) is anHLA-A2402-restricted WT1 peptide, and WAPVLDFAPPGASAYGSL (SEQ ID NO:244) is an MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (5)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (5). Whether or not the helper peptide (SEQID NO: 244) mixed with the formula (5) works in the living body wasjudged by comparison of the number of IFNγ-producing cells when thesplenocytes derived from the above-mentioned mouse administered with acompound represented by the formula (5) alone and the splenocytesderived from the above-mentioned mouse administered with a cocktailvaccine of a compound represented by the formula (5) and the peptideshown by SEQ ID NO: 244 were re-stimulated with the peptide (SEQ ID NO:2).

By a method similar to that in Experimental Example 14, CTL inductiontest was performed. To give a cocktail vaccine, a compound representedby the formula (5) and the peptide shown by SEQ ID NO: 244 weredissolved in dimethyl sulfoxide (DMSO) at 80 mg/mL, diluted with waterfor injection and mixed such that the concentration after dilution was 3mg/mL for the compound represented by the formula (5), and 2.3 mg/mL forthe peptide shown by SEQ ID NO: 244. The diluted solution was mixed withan equal amount of Montanide ISA51VG to give an emulsion. A cocktailvaccine containing the compound represented by the formula (5) at 150μg/site, and the peptide shown by SEQ ID NO: 244 at 115 μg/site wasintradermally administered to 2 sites at the base of tail of a mouse.

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 24.

In FIG. 24, the vertical axis shows the number of cells that reacted inthe plated cells. In FIG. 24, the black bar and the white bar show theresults of culture of HLA-A0201 transgenic mouse-derived splenocytes inthe presence or absence of the object peptide represented by SEQ ID NO:2. That is, the difference in the values of the black bar and the whitebar shows the number of the object, each peptide-specific CTL induced inthe mouse in vivo by the administration of a cocktail vaccine containinga compound represented by the formula (5) and a helper peptide (SEQ IDNO: 244).

In FIG. 24, the value of the white bar is not detected. This means thatthe splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered with acompound represented by the formula (5) alone, and a cocktail vaccinecontaining a helper peptide (SEQ ID NO: 244). In FIG. 24, the number ofthe IFNγ producing cells specific to the peptide shown by SEQ ID NO: 2,which was induced by the administration of a cocktail vaccine containinga helper peptide (SEQ ID NO: 244), was higher than the number of thepeptide-specific IFNγ producing cells, which was induced by theadministration of a compound represented by the formula (5) alone.

From the above, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the peptide shown by SEQ IDNO: 244 can induce CTL specific to the peptides shown by SEQ ID NO: 2.In addition, many IFNγ-producing cells specific to the peptide shown bySEQ ID NO: 2 were found when a cocktail vaccine was administered ascompared to the single administration of a compound represented by theformula (5). It was assumed that the induction of the cell reactive withthe helper peptide shown by SEQ ID NO: 244 contained in the cocktailvaccine enhanced induction of CTL specific to the peptide shown by SEQID NO: 2. Therefore, it was clarified that a cocktail vaccine containinga compound represented by the formula (5) and the helper peptide canstrongly induce CTL in the body of mouse as compared to the singleadministration of a compound represented by the formula (5).

As one embodiment of producing a vaccine containing two WT1 antigenpeptides, a cocktail vaccine containing two different peptides as asingle preparation can be mentioned. When producing a cocktail vaccine,the properties of the cancer antigen peptides to be mixed poses oneproblem. As shown in Table 60 and Table 66, production of a cocktail oftwo WT1 antigen peptides means processing of two peptides havingdifferent solubility, namely, property, into one preparation. Incontrast, the conjugate of the present invention is a compound whereintwo WT1 antigen peptides are bonded via a disulfide bond, and shows asingle solubility, namely, property. This means that the conjugate ofthe present invention has single property and also has the propertycorresponding to the two WT1 antigen peptides, as shown in ExperimentalExample 2. In this aspect, it was shown that the conjugate of thepresent invention is a compound capable of inducing a response to thetwo WT1 antigen peptides without the need to consider an interactionbetween the two WT1 antigen peptides and the like, unlike cocktailvaccines.

Experimental Example 16 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse

The CTL induction ability of a cocktail vaccine of a compoundrepresented by the formula (5) synthesized in Example 1 and the peptideshown by SEQ ID NO: 24 synthesized in Reference Example 2 was evaluatedby an in vivo CTL induction test using an HLA-A0201 transgenic mouse.RMFPNAPYL (SEQ ID NO: 2) contained in a compound represented by theformula (5):

wherein the bond between C and C is a disulfide bond, is anHLA-A0201-restricted WT1 peptide, CYTWNQMNL (SEQ ID NO: 4) is anHLA-A2402-restricted WT1 peptide, and CNKRYFKLSHLQMHSRKTG (SEQ ID NO:24) is an MHC class II-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (5)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (5). Whether or not the helper peptide (SEQID NO: 24) mixed with the formula (5) works in the living body wasjudged by comparison of the number of IFNγ-producing cells when thesplenocytes derived from the above-mentioned mouse administered with acompound represented by the formula (5) alone and the splenocytesderived from the above-mentioned mouse administered with a cocktailvaccine of a compound represented by the formula (5) and the peptideshown by SEQ ID NO: 24 were re-stimulated with the peptide (SEQ ID NO:2).

Specifically, a compound represented by the formula (5) was dissolved indimethyl sulfoxide (DMSO) at 80 mg/mL, further diluted with water forinjection to 3 mg/mL, and emulsified by mixing with an equal amount ofMontanide ISA51VG. The emulsified compound was intradermallyadministered to 2 sites at the base of tail of a mouse at 150 μg/site.In addition, a compound represented by the formula (5) and the peptideshown by SEQ ID NO: 24 were dissolved in dimethyl sulfoxide (DMSO) at 80mg/mL, and diluted with water for injection. They were mixed such thatthe concentration after dilution is 3 mg/mL for the compound representedby the formula (5), and 3.11 mg/mL for the peptide shown by SEQ ID NO:24. The diluted solution was mixed with an equal amount of MontanideISA51VG to give an emulsion. The cocktail vaccine containing a compoundrepresented by the formula (5) at 150 μg/site, and the peptide shown bySEQ ID NO: 24 at 156 μg/site was intradermally administered to 2 sitesat the base of tail of a mouse. The DMSO concentration of each emulsionwas set to the same level. One week later, the mouse was euthanized withCO₂ gas, the spleen was isolated, and splenocytes were prepared. IFNγELISPOT assay kit was used for the measurement of IFNγ production. Onthe previous day of splenocyte preparation, an ELISPOT plate was treatedwith an anti-mouse IFNγ antibody, and blocked with RPMI1640 mediumcontaining 10% FBS the next day. The prepared HLA-A0201 transgenicmouse-derived splenocytes were plated at 0.25×10⁶ cells/well on theblocked ELISPOT plate. Peptide (SEQ ID NO: 2) was dissolved in DMSO at40 mg/mL, and further diluted with RPMI1640 medium containing 10% FBS to40 μg/mL. The diluted peptide (SEQ ID NO: 2) was added to the HLA-A0201transgenic mouse-derived splenocytes at a final concentration of 10μg/mL. The splenocytes added with the peptide were cultured for 19 hr at37° C., 5% CO₂, whereby peptide re-stimulation in vitro was performed.After culture, the supernatant was removed, and the ELISPOT plate wasallowed to develop color according to the attached protocol. The numberof spots that developed color was measured by ImmunoSpot Analyzer(manufactured by C.T.L.).

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 25.

In FIG. 25, the vertical axis shows the number of cells that reacted inthe plated cells. In FIG. 25, the black bar and the white bar show theresults of culture of HLA-A0201 transgenic mouse-derived splenocytes inthe presence or absence of the object peptide represented by SEQ ID NO:2. That is, the difference in the values of the black bar and the whitebar shows the number of the object, each peptide-specific CTL induced inthe mouse in vivo by the administration of a cocktail vaccine containinga compound represented by the formula (5) and a helper peptide (SEQ IDNO: 24).

In FIG. 25, the value of the white bar is not detected. This means thatthe splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered with acompound represented by the formula (5) alone, and a cocktail vaccinecontaining a helper peptide (SEQ ID NO: 24). In FIG. 25, the number ofthe IFNγ producing cells specific to the peptide shown by SEQ ID NO: 2,which was induced by the administration of a cocktail vaccine containinga helper peptide (SEQ ID NO: 24), was higher than the number of thepeptide-specific IFNγ producing cells, which was induced by the singleadministration of a compound represented by the formula (5) alone.

From the above, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the peptide shown by SEQ IDNO: 24 can induce CTL specific to the peptides shown by SEQ ID NO: 2. Inaddition, many IFNγ-producing cells specific to the peptide shown by SEQID NO: 2 were found when a cocktail vaccine was administered as comparedto the single administration of a compound represented by the formula(5). It was assumed that the induction of the cell reactive with thehelper peptide shown by SEQ ID NO: 24 contained in the cocktail vaccineenhanced induction of CTL specific to the peptide shown by SEQ ID NO: 2.Therefore, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the helper peptide canstrongly induce CTL in the body of mouse as compared to the singleadministration of a compound represented by the formula (5).

Experimental Example 17 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse

SEQ ID NO: 242 synthesized in Example 11 is the peptide shown by SEQ IDNO: 244 having an extended cysteine at the N-terminal. SEQ ID NO: 244 inthe cocktail vaccine in Experimental Example 15 shows a CTL inductionenhancing activity. In this test, therefore, the CTL induction abilityof a cocktail vaccine of a compound represented by the formula (5)synthesized in Example 1 and the peptide shown by SEQ ID NO: 242 wasevaluated by an in vivo CTL induction test using an HLA-A0201 transgenicmouse. RMFPNAPYL (SEQ ID NO: 2) contained in a compound represented bythe formula (5):

wherein the bond between C and C is a disulfide bond, is anHLA-A0201-restricted WT1 peptide, CYTWNQMNL (SEQ ID NO: 4) is anHLA-A2402-restricted WT1 peptide, and WAPVLDFAPPGASAYGSL (SEQ ID NO:244) contained in CWAPVLDFAPPGASAYGSL (SEQ ID NO: 242) is an MHC classII-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (5)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (5). Whether or not the helper peptide (SEQID NO: 242) mixed with the formula (5) works in the living body wasjudged by comparison of the number of IFNγ-producing cells when thesplenocytes derived from the above-mentioned mouse administered with acompound represented by the formula (5) alone and the splenocytesderived from the above-mentioned mouse administered with a cocktailvaccine of a compound represented by the formula (5) and the peptideshown by SEQ ID NO: 242 were re-stimulated with the peptide (SEQ ID NO:2).

By a method similar to that in Experimental Example 16, a CTL inductiontest was performed. To give a cocktail vaccine, a compound representedby the formula (5) and the peptide shown by SEQ ID NO: 242 weredissolved in dimethyl sulfoxide (DMSO) at 80 mg/mL, diluted with waterfor injection and mixed such that the concentration after dilution was 3mg/mL for the compound represented by the formula (5), and 2.42 mg/mLfor the peptide shown by SEQ ID NO: 242. The diluted solution was mixedwith an equal amount of Montanide ISA51VG to give an emulsion. Acocktail vaccine containing the compound represented by the formula (5)at 150 μg/site, and the peptide shown by SEQ ID NO: 242 at 121 μg/sitewas intradermally administered to 2 sites at the base of tail of amouse.

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 26.

In FIG. 26, the vertical axis shows the number of cells that reacted inthe plated cells. In FIG. 26, the black bar and the white bar show theresults of culture of HLA-A0201 transgenic mouse-derived splenocytes inthe presence or absence of the object peptide represented by SEQ ID NO:2. That is, the difference in the values of the black bar and the whitebar shows the number of the object, each peptide-specific CTL induced inthe mouse in vivo by the administration of a cocktail vaccine containinga compound represented by the formula (5) and a helper peptide (SEQ IDNO: 242).

In FIG. 26, the value of the white bar is not detected. This means thatthe splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered with acompound represented by the formula (5) alone, and a cocktail vaccinecontaining a helper peptide (SEQ ID NO: 242). In FIG. 26, the number ofthe IFNγ producing cells specific to the peptide shown by SEQ ID NO: 2,which was induced by the administration of a cocktail vaccine containinga helper peptide (SEQ ID NO: 242), was higher than the number of thepeptide-specific IFNγ producing cells, which was induced by theadministration of a compound represented by the formula (5) alone.

From the above, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the peptide shown by SEQ IDNO: 242 can induce CTL specific to the peptides shown by SEQ ID NO: 2.In addition, many IFNγ-producing cells specific to the peptide shown bySEQ ID NO: 2 were found when a cocktail vaccine was administered ascompared to the single administration of a compound represented by theformula (5). It was assumed that the induction of the cell reactive withthe helper peptide shown by SEQ ID NO: 244 contained in the peptideshown by SEQ ID NO: 242 contained in the cocktail vaccine enhancedinduction of CTL specific to the peptide shown by SEQ ID NO: 2.Therefore, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the helper peptide canstrongly induce CTL in the body of mouse as compared to the singleadministration of a compound represented by the formula (5).

Experimental Example 18 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A0201 Transgenic Mouse

SEQ ID NO: 243 synthesized in Example 12 is the peptide shown by SEQ IDNO: 244 having an extended cysteine at the N-terminal. SEQ ID NO: 244 inthe cocktail vaccine in Experimental Example 15 shows a CTL inductionenhancing activity. In this test, therefore, the CTL induction abilityof a cocktail vaccine of a compound represented by the formula 1.5 (5)synthesized in Example 1 and the peptide shown by SEQ ID NO: 243 wasevaluated by an in vivo CTL induction test using an HLA-A0201 transgenicmouse. RMFPNAPYL (SEQ ID NO: 2) contained in a compound represented bythe formula (5):

wherein the bond between C and C is a disulfide bond, is anHLA-A0201-restricted WT1 peptide, CYTWNQMNL (SEQ ID NO: 4) is anHLA-A2402-restricted WT1 peptide, and WAPVLDFAPPGASAYGSL (SEQ ID NO:244) contained in WAPVLDFAPPGASAYGSLC (SEQ ID NO: 243) is an MHC classII-restricted WT1 peptide (namely, helper peptide).

The HLA-A0201 transgenic mouse is as described in Experimental Examples2 and 5.

Whether the administration of a compound represented by the formula (5)results in the induction of CTL specific to the object peptide (SEQ IDNO: 2) was judged based on the measurement of IFNγ production byre-stimulation, with the peptide (SEQ ID NO: 2), of the splenocytederived from the above-mentioned mouse administered with a compoundrepresented by the formula (5). Whether or not the helper peptide (SEQID NO: 243) mixed with the formula (5) works in the living body wasjudged by comparison of the number of IFNγ-producing cells when thesplenocytes derived from the above-mentioned mouse administered with acompound represented by the formula (5) alone and the splenocytesderived from the above-mentioned mouse administered with a cocktailvaccine of a compound represented by the formula (5) and the peptideshown by SEQ ID NO: 243 were re-stimulated with the peptide (SEQ ID NO:2).

By a method similar to that in Experimental Example 16, a CTL inductiontest was performed. To give a cocktail vaccine, a compound representedby the formula (5) and the peptide shown by SEQ ID NO: 243 weredissolved in dimethyl sulfoxide (DMSO) at 80 mg/mL, diluted with waterfor injection and mixed such that the concentration after dilution was 3mg/mL for the compound represented by the formula (5), and 2.42 mg/mLfor the peptide shown by SEQ ID NO: 243. The diluted solution was mixedwith an equal amount of Montanide ISA51VG to give an emulsion. Acocktail vaccine containing the compound represented by the formula (5)at 150 μg/site, and the peptide shown by SEQ ID NO: 243 at 121 μg/sitewas intradermally administered to 2 sites at the base of tail of amouse.

The results of IFNγ ELISPOT assay using the HLA-A0201 transgenic mouseare shown in FIG. 27.

In FIG. 27, the vertical axis shows the number of cells that reacted inthe plated cells. In FIG. 27, the black bar and the white bar show theresults of culture of HLA-A0201 transgenic mouse-derived splenocytes inthe presence or absence of the object peptide represented by SEQ ID NO:2. That is, the difference in the values of the black bar and the whitebar shows the number of the object, each peptide-specific CTL induced inthe mouse in vivo by the administration of a cocktail vaccine containinga compound represented by the formula (5) and a helper peptide (SEQ IDNO: 243).

In FIG. 27, the value of the white bar is not detected. This means thatthe splenocytes of respective transgenic mice did not react in theabsence of the object peptide. As a result of this test, IFNγ productionspecific to the object peptide shown by SEQ ID NO: 2 was detected in thesplenocytes derived from HLA-A0201 transgenic mouse administered with acompound represented by the formula (5) alone, and a cocktail vaccinecontaining a helper peptide (SEQ ID NO: 243). In FIG. 27, the number ofthe IFNγ producing cells specific to the peptide shown by SEQ ID NO: 2,which was induced by the administration of a cocktail vaccine containinga helper peptide (SEQ ID NO: 243), was higher than the number of thepeptide-specific IFNγ producing cells, which was induced by theadministration of a compound represented by the formula (5) alone.

From the above, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the peptide shown by SEQ IDNO: 243 can induce CTL specific to the peptides shown by SEQ ID NO: 2.In addition, many IFNγ-producing cells specific to the peptide shown bySEQ ID NO: 2 were found when a cocktail vaccine was administered ascompared to the single administration of a compound represented by theformula (5). It was assumed that the induction of the cell reactive withthe helper peptide shown by SEQ ID NO: 244 contained in the peptideshown by SEQ ID NO: 243 contained in the cocktail vaccine enhancedinduction of CTL specific to the peptide shown by SEQ ID NO: 2.Therefore, it was clarified that a cocktail vaccine containing acompound represented by the formula (5) and the helper peptide canstrongly induce CTL in the body of mouse as compared to the singleadministration of a compound represented by the formula (5).

As one embodiment of producing a vaccine containing two WT1 antigenpeptides, a cocktail vaccine containing two different peptides as asingle preparation can be mentioned. When producing a cocktail vaccine,the properties of the cancer antigen peptides to be mixed poses oneproblem. As shown in Table 60 and Table 66, production of a cocktail oftwo WT1 antigen peptides means processing of two peptides havingdifferent solubility, namely, property, into one preparation. Incontrast, the conjugate of the present invention is a compound whereintwo WT1 antigen peptides are bonded via a disulfide bond, and shows asingle solubility, namely, property. This means that the conjugate ofthe present invention has single property and also has the propertycorresponding to the two WT1 antigen peptides, as shown in ExperimentalExample 2. In this aspect, it was shown that the conjugate of thepresent invention is a compound capable of inducing a response to thetwo WT1 antigen peptides without the need to consider an interactionbetween the two WT1 antigen peptides and the like, unlike cocktailvaccines.

Experimental Example 19 Evaluation of In Vivo CTL Induction AbilityUsing HLA-A2402 Transgenic Mouse after Filter Filtration

The homodimer shown by SEQ ID NO: 4 formed via a disulfide bond and acompound represented by the formula (5) are dissolved in water forinjection at 3-10 mg/mL. The pharmacological activity of each compoundis evaluated using an HLA-A2402 transgenic mouse (C57BL/6CrHLA-A2402/e)with the CTL induction activity as an index. For administration to theHLA-A2402 transgenic mouse, the compound is dissolved in water forinjection, sterilized by filtration using a low protein-binding filter(membrane filter of the grade aiming at sterilization treatment ofinjection) and mixed with incomplete Freund's adjuvant to give anemulsion.

The emulsified compound is intradermally administered to the tail rootof an HLA-A2402 transgenic mouse. One week later, the mouse iseuthanized with CO₂ gas, the spleen or inguinal lymph node is isolated,and splenocytes or or lymph node cells are prepared. IFNγ ELISPOT assaykit is used for the measurement of IFNγ production. On the previous dayof cell preparation, an ELISPOT plate is treated with an anti-mouse IFNγantibody, and blocked with RPMI1640 medium containing 10% FBS the nextday. The prepared mouse-derived cells are plated on the blocked ELISPOTplate. Peptide (SEQ ID NO: 4) is dissolved in DMSO at 40 mg/mL, andfurther diluted with RPMI1640 medium containing 10% FBS to 40 μg/mL. Thediluted peptide (SEQ ID NO: 4) is added to the HLA-A2402 transgenicmouse-derived splenocytes or lymph node cells at a final concentrationof 10 μg/mL. The cells added with the peptide are cultivated for 16-20hr at 37° C., 5% CO₂, whereby peptide re-stimulation in vitro isperformed. After culture, the supernatant is removed, and the ELISPOTplate is allowed to develop color according to the attached protocol.The number of spots that developed color is measured by ImmunoSpotAnalyzer (manufactured by C.T.L.).

INDUSTRIAL APPLICABILITY

The compound of the present invention is useful as an active ingredientof a cancer vaccine that efficiently induces CTL and is easy to produce.This application is based on a patent application Nos. 2013-072173(filing date: Mar. 29, 2013) and 2013-158383 filed in Japan (filingdate: Jul. 31, 2013), the whole contents of which are incorporated intothis specification.

The invention claimed is:
 1. A method of treating WT1 positive cancer,comprising: administering a therapeutically effective amount of apharmaceutical composition to a WT1 positive cancer patient in needthereof, the pharmaceutical composition comprising a compound selectedfrom the group consisting of

wherein the bond between CR is a bond between the carbonyl group of Cand the N-terminal amino group of R, and the bond between C and C is adisulfide bond, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 2. The method of claim 1, whereinthe pharmaceutical composition comprises the compound having the formula(5):

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein the pharmaceutical composition comprises the compound having theformula (5):

and a peptide consisting of an amino acid sequence: CNKRYFKLSHLQMHSRK.(SEQ ID NO: 22) 


4. The method of claim 1, wherein the pharmaceutical compositioncomprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence:CNKRYFKLSHLQMHSRKHTG. (SEQ ID NO: 24)


5. The method of claim 1, wherein the pharmaceutical compositioncomprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence: CWAPVLDFAPPGASAYGSL.(SEQ ID NO: 242)


6. The method of claim 1, wherein the pharmaceutical compositioncomprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence: WAPVLDFAPPGASAYGSLC.(SEQ ID NO: 243)


7. The method of claim 1, wherein the pharmaceutical compositioncomprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence: WAPVLDFAPPGASAYGSL.(SEQ ID NO: 244)


8. The method of claim 1, wherein the WT1 positive cancer is one ofleukemia, myelodysplastic syndrome, multiple myeloma, malignantlymphoma, gastric cancer, colorectal cancer, lung cancer, breast cancer,germ cell cancer, liver cancer, skin cancer, urinary bladder cancer,prostate cancer, uterine cancer, cervical cancer, ovarian cancer, andbrain tumor.
 9. The method of claim 1, wherein the pharmaceuticalcomposition comprises a peptide consisting of an amino acid sequence:CNKRYFKLSHLQMHSRK. (SEQ ID NO: 22) 


10. The method of claim 1, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:CNKRYFKLSHLQMHSRKHTG. (SEQ ID NO: 24)


11. The method of claim 1, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:CWAPVLDFAPPGASAYGSL. (SEQ ID NO: 242)


12. The method of claim 1, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:WAPVLDFAPPGASAYGSLC. (SEQ ID NO: 243)


13. The method of claim 1, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:WAPVLDFAPPGASAYGSL. (SEQ ID NO: 244)


14. The method of claim 1, wherein the pharmaceutical compositioncomprises the compound having the formula (4):

or a pharmaceutically acceptable salt thereof.
 15. A method of inducingcytotoxic T cells against WT1 positive cancer, comprising: administeringan effective amount of a pharmaceutical composition to a subject, thepharmaceutical composition comprising a compound selected from the groupconsisting of

wherein the bond between CR is a bond between the carbonyl group of Cand the N-terminal amino group of R, and the bond between C and C is adisulfide bond, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 16. The method of claim 15, whereinthe pharmaceutical composition comprises the compound having the formula(5):

or a pharmaceutically acceptable salt thereof.
 17. The method of claim15, wherein the pharmaceutical composition comprises the compound havingthe formula (5):

and a peptide consisting of an amino acid sequence: CNKRYFKLSHLQMHSRK.(SEQ ID NO: 22)


18. The method of claim 15, wherein the pharmaceutical compositioncomprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence: CNKRYFKLSHLQMHSRKHTG(SEQ ID NO: 24).
 19. The method of claim 10, wherein the pharmaceuticalcomposition comprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence: CWAPVLDFAPPGASAYGSL.(SEQ ID NO: 242)


20. The method of claim 15, wherein the pharmaceutical compositioncomprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence: WAPVLDFAPPGASAYGSLC.(SEQ ID NO: 243)


21. The method of claim 15, wherein the pharmaceutical compositioncomprises the compound having the formula (5):

and a peptide consisting of an amino acid sequence: WAPVLDFAPPGASAYGSL.(SEQ ID NO: 244)


22. The method of claim 15, wherein the WT1 positive cancer is one ofleukemia, myelodysplastic syndrome, multiple myeloma, malignantlymphoma, gastric cancer, colorectal cancer, lung cancer, breast cancer,germ cell cancer, liver cancer, skin cancer, urinary bladder cancer,prostate cancer, uterine cancer, cervical cancer, ovarian cancer, andbrain tumor.
 23. The method of claim 15, wherein the pharmaceuticalcomposition comprises a peptide consisting of an amino acid sequence:CNKRYFKLSHLQMHSRK. (SEQ ID NO: 22)


24. The method of claim 15, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:CNKRYFKLSHLQMHSRKHTG. (SEQ ID NO: 24)


25. The method of claim 15, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:CWAPVLDFAPPGASAYGSL. (SEQ ID NO: 242)


26. The method of claim 15, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:WAPVLDFAPPGASAYGSLC. (SEQ ID NO: 243)


27. The method of claim 15, wherein the pharmaceutical compositioncomprises a peptide consisting of an amino acid sequence:WAPVLDFAPPGASAYGSL. (SEQ ID NO: 244)


28. The method of claim 15, wherein the pharmaceutical compositioncomprises the compound having the formula (4):

or a pharmaceutically acceptable salt thereof.